1 short title: pectin elongation requires muci70 and ... · 9/18/2018 · are further branched...
TRANSCRIPT
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Short Title Pectin Elongation Requires MUCI70 and GAUT11 1
2
Cătălin Voiniciuc 3
Institute for Bio- and Geosciences (IBG-2 Plant Sciences) Forschungszentrum Juumllich 52425 4
Juumllich Germany 5
Current Address Institute for Plant Cell Biology and Biotechnology Heinrich Heine University 6
40225 Duumlsseldorf Germany 7
+49 211 81-15996 8
catalinvoiniciuchhude 9
10
RESEARCH AREA Biochemistry and Metabolism 11
12
13
Plant Physiology Preview Published on September 18 2018 as DOI101104pp1800584
Copyright 2018 by the American Society of Plant Biologists
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Identification of Key Enzymes for Pectin Synthesis in Seed Mucilage 14
Cătălin Voiniciuc 1 Kristen A Engle1 Markus Guumlnl Sabine Dieluweit Maximilian Heinrich-15
Wilhelm Schmidt Jeong-Yeh Yang Kelley W Moremen Debra Mohnen and Bjoumlrn Usadel 16
17 Institute for Bio- and Geosciences (IBG-2 Plant Sciences) Forschungszentrum Juumllich 52425 18
Juumllich Germany (CV MH-WS MG BU) Institute for Botany and Molecular Genetics 19
(IBMG) BioSC RWTH Aachen University 52074 Aachen Germany (CV MH-WS BU) 20
Department of Biochemistry and Molecular Biology (KWM DM) Department of Plant Biology 21
(KE) University of Georgia Athens Georgia USA (KAE J-YY KWMDM) Complex 22
Carbohydrate Research Center University of Georgia Athens Georgia USA (KAE J-YY 23
KWM DM) 24
Institute of Complex Systems (ICS-7) Forschungszentrum Juumllich 52425 Juumllich Germany (SD) 25
26
E-Mails catalinvoiniciuchhude (CV) kengleugaedu (KAE) mguenlfz-juelichde 27
(MG) sdieluweitfz-juelichde (SD) mschmidtfz-juelichde (MH-WS) jyyangugaedu 28
(JYY) moremenugaedu (KWM) dmohnenccrcugaedu (DM) usadelbio1rwth-29
aachende (BU) 30
31
One-Sentence Summary 32
Mutations in two glycosyltransferase-encoding genes severely impair the elongation of pectic 33
rhamnogalacturonan I resulting in hydrophobic seeds that do not release mucilage polymers 34
35
FOOTNOTES 36
Author Contributions 37
CV designed research and wrote the article with valuable input from BU MG analyzed 38
glycosyl linkages and assisted with HPAEC-PAD work SB performed SEM analysis MS 39
cloned MUCI70 in E coli KAE and J-YY designed and carried out the GAUT11 expression 40
and enzyme analysis research with advice from KWM and DM CV performed the remaining 41
experiments All authors read the article provided comments and approved the final version 42
Funding Information 43
The research was supported by the Natural Sciences and Engineering Research Council of 44
Canada (NSERC PGS-D3 to CV) Deutsche Forschungsgemeinschaft (US9813-1) and by the 45
Ministry of Innovation Science and Research of North-Rhine Westphalia within the framework 46
of the NRW Strategieprojekt BioSC (No 313323‐400‐00213 to MH-WS and BU) The 47
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research was also supported by BioEnergy Science Center Grant (DE-PS02-06ER64304) and 48
the Center for Bioenergy Innovation The BioEnergy Science Center and the Center for 49
Bioenergy Innovation are US Department of Energy Bioenergy Research Centers supported 50
by the Office of Biological and Environmental Research in the Department of Energyrsquos Office of 51
Science The research was also partially funded by the Department of Energy Center Grant DE-52
SC0015662 and US National Institutes of Health grants P41GM103390 and P01GM107012 53
Generation of the CCRC series of monoclonal antibodies used in this work was supported by a 54
grant from the NSF Plant Genome Program (DBI-0421683) 55
Present Address 56
Institute for Plant Cell Biology and Biotechnology Heinrich Heine University 40225 Duumlsseldorf 57
Germany (CV) 58
Address correspondence to catalinvoiniciuchhude 59 60 1 These authors contributed equally to the experimental work 61 62 The author responsible for distribution of materials integral to the findings presented in this 63
article in accordance with the policy described in the Instructions for Authors 64
(wwwplantphysiolorg) is Cătălin Voiniciuc (catalinvoiniciuchhude) 65
ABSTRACT 66
Pectin is a vital component of the plant cell wall and provides the molecular glue that maintains 67
cell-cell adhesion among other functions As the most complex wall polysaccharide pectin is 68
composed of several covalently-linked domains such as homogalacturonan (HG) and 69
rhamnogalacturonan I (RG I) Pectin has widespread uses in the food industry and has 70
emerging biomedical applications but its synthesis remains poorly understood For instance 71
the enzymes that catalyze RG I elongation remain unknown Recently a co-expression and 72
sequence-based MUCILAGE-RELATED (MUCI) reverse genetic screen uncovered 73
hemicellulose biosynthetic enzymes in the Arabidopsis thaliana seed coat Here we use an 74
extension of this strategy to identify MUCI70 as the founding member of a glycosyltransferase 75
family essential for the accumulation of seed mucilage a gelatinous wall rich in unbranched RG 76
I Detailed biochemical and histological characterization of two muci70 mutants and two gaut11 77
mutants identified MUCI70 and GAUT11 as required for two distinct RG I domains in seed 78
mucilage We demonstrate that unlike MUCI70 GAUT11 catalyzes HG elongation in vitro and 79
is thus likely required for the synthesis of an HG region important for RG I elongation Analysis 80
of a muci70 gaut11 double mutant confirmed that MUCI70 and GAUT11 are indispensable for 81
the production and release of the bulk of mucilage RG I and for shaping the surface 82
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morphology of seeds In addition we uncover relationships between pectin and hemicelluloses 83
and show that xylan is essential for the elongation of at least one RG I domain 84
85
INTRODUCTION 86
Plant cell walls are largely composed of three major classes of polysaccharides 87
cellulose hemicellulose and pectin While cellulose and hemicelluloses are largely built of 88
neutral sugars connected by β-14-linkages pectin is defined by its high content of galacturonic 89
acid (GalA) residues connected by α-14-linkages Cellulose-hemicellulose networks have been 90
thought to provide the tensile strength of the wall while pectin was mainly implicated in cell-cell 91
adhesion and determining the porosity of the wall (Cosgrove 2016) However recent evidence 92
indicates that pectin-cellulose junctions are more prevalent than previously expected and thus 93
that pectin may play additional structural roles (Wang et al 2015) Furthermore since 94
mutations in several pectin-related genes are lethal it is evident that this matrix polysaccharide 95
has vital functions in plants (Caffall et al 2009) Pectin also has widespread uses in the food 96
industry and has emerging applications in the biomedical field including use as a gelling agent 97
for targeted drug delivery and as a bioactive molecule for cancer treatment (Maxwell et al 98
2012 Munarin et al 2012) 99
Pectin is the most complex polysaccharide in the plant cell wall consisting of multiple 100
glycan domains that may exist in one or more polymers linked via their backbones (Nakamura 101
et al 2002 Atmodjo et al 2013) The backbone of the most abundant extractable pectin 102
consists exclusively of D-GalA subunits and can be unbranched (Homogalacturonan HG) 103
substituted with D-xylose (Xyl) residues (Xylogalacturonan) or decorated with a conserved set 104
of side chains (Rhamnogalacturonan II RG II) In contrast the backbone of 105
Rhamnogalacturonan I (RG I) consists of a repeating α-D-14-GalA-α-L-12-Rha disaccharide 106
The rhamnose (Rha) residues in the RG I backbone can be frequently substituted with a wide 107
variety of oligosaccharide or polysaccharide side chains Around 40 different RG I side chain 108
structures have been reported so far (Atmodjo et al 2013) including linear β-14-linked D-109
galactan and α-15-linked L-arabinan or arabinogalactans containing both galactose (Gal) and 110
arabinose (Ara) units Despite the biochemical evidence that HG and RG I are covalently linked 111
in soybean (Nakamura et al 2002) the full in vivo structure of the pectin macromolecules has 112
yet to be determined due to the difficulty of extracting them in an intact form (Atmodjo et al 113
2013) In addition a complex proteoglycan purified from Arabidopsis (Arabidopsis thaliana) 114
suspension cultures has been shown to contain covalently linked HG and RG I domains which 115
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are further branched with the hemicellulose xylan (Tan et al 2013) This finding suggests that 116
certain pectin domains such as RG I may have a more central role in cell wall organization than 117
previously thought 118
Based on the large number of pectin structures that have been detected in plants their 119
biosynthesis is hypothesized to require at least 67 distinct enzymes that transfer glycosyl 120
methyl or acetyl groups (Atmodjo et al 2013) However only four types of pectin biosynthetic 121
enzymes have been identified and biochemically characterized so far These include 122
glycosyltransferase (GT) proteins that belong to four different Carbohydrate-Active Enzyme 123
(CAZy httpwwwcazyorg Lombard et al 2014) families GT8 GT47-C GT77 and GT92 124
Two GT8 proteins GALACTURONOSYLTRANSFERASE1 (GAUT1) and GAUT7 form the core 125
of a GAUT1GAUT7 complex that catalyzes the elongation of the HG backbone (Sterling et al 126
2006 Atmodjo et al 2011) Additional GAUT and GAUT-LIKE (GATL) proteins from the GT8 127
family encode proven and putative HG galacturonosyltransferases (α-GalA transferases) For 128
example GAUT4 was recently shown to be an HG α-GalA transferase whose downregulation 129
results in reduced HG and RG II production (Biswal et al 2018) Although GAUT1 and GAUT7 130
are predicted to have similar protein topologies they have surprisingly distinct functions In vivo 131
the GAUT1 enzyme is cleaved into a soluble form that is retained at the site of pectin synthesis 132
via interactions with GAUT7 a Golgi membrane-bound protein anchor with no demonstrated 133
catalytic activity (Atmodjo et al 2011) Unlike GAUT4 and the GAUT1GAUT7 complex which 134
synthesize the HG backbone the other GTs known to be involved in pectin synthesis catalyze 135
the synthesis of three distinct pectin side chains the β-13-xylosyl branches of xylogalacturonan 136
(GT47-C Jensen et al 2008) the α-13-xylosyl residues in RG II (GT77 Egelund et al 2006) 137
and the β-14-galactan side chains of RG I (GT92 Liwanag et al 2012) Overall these GT 138
activities account for only a small fraction of the pectin structures found in nature In addition 139
there is increasing evidence that seemingly distinct wall polymers such as pectin and the 140
hemicellulose xylan are structurally dependent on one another (Hao and Mohnen 2014) For 141
example the loss of GAUT12 (a GT8 protein) in the irregular xylem8 (irx8) mutant leads to 142
dwarf plants that have significant reductions in both xylan and HG (Pentildea et al 2007 Persson et 143
al 2007) Therefore the production of pectin remains poorly understood on a mechanistic level 144
and most of the molecular players involved in this process remain unknown 145
Although co-expression analysis has been a successful approach to identify GTs 146
involved in cellulose and hemicellulose biosynthesis (Brown et al 2005 Persson et al 2005) it 147
previously failed to predict obvious candidates for pectin production Two potential challenges 148
are that pectin biosynthetic enzymes may lack distinctive expression profiles in most plant 149
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6
tissues and that the relevant GTs are not part of classified CAZy families (Harholt et al 2010) 150
These obstacles were surpassed by the identification of novel GT-like plant proteins through 151
Golgi proteomic studies (Nikolovski et al 2012 Nikolovski et al 2014) and the establishment 152
of Arabidopsis seed mucilage as a model for dissecting pectin synthesis (Haughn and Western 153
2012) Within a narrow developmental window Arabidopsis seed coat epidermal (SCE) cells 154
produce copious amounts of RG I along with minor amounts of cellulose hemicellulose 155
arabinogalactans and HG (Voiniciuc et al 2015c) Since at least 90 of the mucilage 156
extracted from Arabidopsis seeds consists of Rha and GalA units derived from pectin the SCE 157
cells can be exploited to identify pectin-related GTs In addition structural changes in seed 158
mucilage polysaccharides can be conveniently monitored in situ with a variety of imaging 159
techniques and specific probes (Voiniciuc et al 2018) 160
Despite the great potential of this model system only two GTs have been implicated so 161
far in the synthesis of the pectin domains in mucilage A screen of 26 gaut mutant lines for 162
altered staining of seed mucilage found only one mutant (gaut11-2) that showed smaller 163
mucilage capsules and reduced uronic acid content compared to the wild type (Caffall et al 164
2009) Although the results indicated that GAUT11 might affect HG biosynthesis in SCE cells 165
the gaut11-2 phenotype was not supported by an independent knockdown gaut11-1 allele 166
(Caffall et al 2009) GATL5 another protein from the GT8 family is the only other pectin-167
related GT that has been implicated in mucilage biosynthesis A knockout T-DNA insertion in 168
GATL5 increased the molecular weight of mucilage polysaccharides without dramatically 169
altering the glycosidic linkage composition or the content of pectin epitopes bound by antibodies 170
(Kong et al 2013) Since GATL5 was proposed to simply regulate the final size of pectin 171
polymers in mucilage additional players must be required for the elongation of RG I in 172
Arabidopsis SCE cells 173
Recently a co-expression and sequence-based MUCILAGE-RELATED (MUCI) reverse 174
genetic screen identified three GTs required for the synthesis of two distinct hemicellulosic 175
polymers (xylan and galactoglucomannan) in Arabidopsis SCE cells (Voiniciuc et al 2015b 176
Voiniciuc et al 2015a) Using an extension of this strategy we now report that the biosynthesis 177
of pectin requires MUCI70 a putative GT from an unclassified CAZy family that was not known 178
to affect cell wall structure Through a detailed biochemical and histological characterization of 179
muci70 mutants and two novel gaut11 alleles we show that these two genes are required for 180
the production of two distinct RG I domains essential for seed mucilage architecture Finally the 181
analysis of a muci70 gaut11 double mutant and the demonstration that GAUT11 is an HG α-182
GalA transferase confirms that MUCI70 and GAUT11 are indispensable for the production of 183
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two RG I domains that represent the bulk of seed mucilage and shape the surface morphology 184
of seeds 185
186
RESULTS 187
MUCI70 is a Novel Pectin-Related GT Localized in the Golgi apparatus 188
To identify novel players involved in pectin production we systematically profiled the expression 189
of all 1128 Arabidopsis thaliana members of the CAZy database (Lombard et al 2014) in the 190
seed coat using ATH1 microarray data in Genevestigator (Hruz et al 2008) This strategy 191
revealed more than 50 CAZy genes that are transcribed in the seed coat when mucilage is 192
produced The majority of these genes were not identified in the initial MUCI screen (Voiniciuc 193
et al 2015b) because they are not significantly co-expressed with known mucilage genes in 194
GeneCAT (Mutwil et al 2008) GeneMANIA (Warde-Farley et al 2010) and ATTED-II 195
(Obayashi et al 2014) Amongst this collection of genes were MUCI64IRX14 (Voiniciuc et al 196
2015a) and four members of the GAUT family (Supplemental Fig S1) including GAUT11 and 197
GATL5 Interestingly we also found one gene encoding a putative GT (At1g28240) which we 198
named MUCI70 as a promising candidate for pectin production in the Arabidopsis seed coat 199
MUCI70 represents the founding member of a GT family whose roles in cell wall biology 200
remain unclear (Fig 1A) The MUCI70 protein contains a single transmembrane domain 201
(AREMEMNON Consensus TM α-helix prediction AramTmConl Schwacke et al 2003) near its 202
N-terminus and a DUF616 (PF04765) conserved domain of unknown function (Fig 1B) 203
Phylogenetic analysis of DUF616 proteins organized MUCI70 and its six Arabidopsis paralogs 204
into four clades (Fig 1A) Each of these groups contains at least one ortholog in both 205
Physcomitrella patens and Selaginella moellendorffii members of two early diverging lineages 206
of land plants (Fig 1A) In contrast TURGOR REGULATION DEFECT 1 (TOD1 AT5G46220) 207
the only other Arabidopsis protein containing a DUF616 motif did not cluster with any of these 208
clades (Fig 1A) and appeared to be functionally distinct Indeed TOD1 was demonstrated to 209
have alkaline ceramidase activity in vitro (Chen et al 2015) rather than a CAZy-related 210
function At4g38500 a close paralog of MUCI70 (Fig 1A) was previously identified in a Golgi 211
proteomics study and showed little similarity in primary sequence and predicted 3D structure to 212
the GT8 family in Arabidopsis (Nikolovski et al 2012) Based on tight co-expression with GAUT 213
genes At4g38500 was hypothesized to be involved in pectin biosynthesis (Voxeur et al 2012) 214
MUCI70 and GAUT11 a gene that was implicated in mucilage HG biosynthesis (Caffall et 215
al 2009) showed similar transcriptional profiles in developing seeds (Supplemental Fig S1 216
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Belmonte et al 2013) and encode proteins with similar topologies (Fig 1B) GAUT11 was 217
previously found in the Golgi proteome (Parsons et al 2012) but the subcellular localization of 218
MUCI70 remained unknown To address this MUCI70 tagged with super yellow fluorescent 219
protein (sYFP) was stably expressed in Arabidopsis using the constitutive 35S promoter 220
MUCI70-sYFP was observed in intracellular punctae (Fig 1C) that co-localized with the Golgi 221
marker sialyltransferase (ST) tagged with red fluorescent protein (ST-RFP Figs 1D and 1E) 222
which marks the site of pectin production in plants 223
224
Mutations in MUCI70 and GAUT11 Cause Severe Mucilage Defects 225
To investigate the biological role of MUCI70 in SCE cells we obtained two independent T-226
DNA lines and identified homozygous mutants (Fig 2A Supplemental Table S1) While GATL5 227
was unambiguously shown to be required for mucilage pectin structure (Kong et al 2013) only 228
one of two transcriptional knockdown mutants (gaut11-2 Fig 2A) previously indicated that 229
GAUT11 influences mucilage structure (Caffall et al 2009) Therefore we analyzed two muci70 230
insertional mutants alongside two gaut11 mutants gaut11-3 and gaut11-4 with insertions in 231
GAUT11 exons (Fig 2A) Using reverse transcription quantitative polymerase chain reaction 232
(RT-qPCR) we validated that both MUCI70 and GAUT11 were transcribed in developing 233
Arabidopsis siliques from 3 to 10 days post anthesis (DPA) GAUT11 showed a dramatic 234
increase (around 15-fold) in expression at 7 DPA when pectin synthesis in SCE cells is at its 235
peak (Fig 2B) Based on the results of public microarray datasets (Winter et al 2007 Belmonte 236
et al 2013) both genes were preferentially expressed in the seed coat relative to the embryo 237
(Fig 2C) and had similar transcript levels from the heart stage (~3 DPA) to the mature green 238
stage (~10 DPA) Each insertion in the MUCI70 gene reduced its expression by at least 60 239
(Fig 2D) Although gaut11-3 and gaut11-4 did not significantly alter GAUT11 transcription at 240
either the 5 or 3 end (Fig 2D) these alleles and the previously described gaut11-2 (Caffall et 241
al 2009) are exonic insertions (Fig 2A) that likely disrupt the GAUT11 protein sequence 242
In contrast to wild-type seeds which are surrounded by large mucilage capsules (Fig 3A) 243
two muci70 and two gaut11 homozygous mutants showed severe ruthenium red (RR) staining 244
defects (Fig 3B to 3E) consisting of patchy or completely impaired mucilage release 245
Consequently the muci70-1 muci70-2 and gaut11-3 seeds were surrounded by significantly 246
smaller mucilage capsules (Fig 2E) whose surface area was only 19 to 39 of the wild-type 247
value At least 65 of muci70 and gaut11 seeds floated on water (Fig 2F Fig 3 marked by 248
stars) whereas wild-type seeds did not float (Fig 2F) despite having similar dimensions (Fig 249
2E) Besides GAUT11 three other GAUT genes (GAUT8 GAUT10 GAUT14) were expressed 250
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9
in the developing seed coat (Supplemental Fig S1) The gaut8 mutant was previously found to 251
be lethal but the gaut10-1 and gaut14-1 transcriptional knockout mutants were viable (Caffall et 252
al 2009) and re-examined in this study In contrast to muci70 and gaut11 mutants gaut10-1 253
and gaut14-1 did not disrupt the dimensions of the seeds or the surrounding RR-stained 254
mucilage capsules (Fig 2E Supplemental Fig S2) Therefore only one of the GAUT genes 255
tested was essential on its own for maintaining mucilage architecture consistent with the 256
previous study of the whole GAUT family (Caffall et al 2009) 257
Since both gaut11-3 and gaut11-4 mutants showed similar mucilage staining defects to the 258
previously described gaut11-2 allele (Caffall et al 2009) we primarily used gaut11-3 for further 259
experiments To investigate if MUCI70 and GAUT11 function in the same pathway we crossed 260
the muci70-1 and gaut11-3 single mutants While muci70 and gaut11 single mutants showed 261
smaller RR-stained mucilage capsules than the wild type (Fig 3A to 3E) all muci70 gaut11 262
double mutant seeds failed to release mucilage (Fig 3F) and thus floated on water (Fig 2F) 263
Despite the severe mucilage defects the muci70 gaut11 seeds were only 6 smaller than the 264
wild type (Fig 2E) This suggested that both MUCI70 and GAUT11 might be required for the 265
biosynthesis of pectin in SCE cells which is ultimately released as a hydrophilic capsule from 266
mature seeds 267
268
MUCI70 and GAUT11 Are Important for Pectin Production in SCE Cells 269
To identify the underlying biochemical defects that lead to impaired mucilage release total 270
mucilage was extracted from seeds vigorously mixed using a ball mill (Voiniciuc et al 2015b 271
Voiniciuc and Guumlnl 2016) As previously described this intensive mechanical agitation 272
effectively removes all mucilage polysaccharides resulting in seeds that are no longer stained 273
by RR (Fig 4A) The monosaccharide composition of the total mucilage extracted from hydrated 274
seeds was quantified using high-performance anion-exchange chromatography with pulsed 275
amperometric detection (HPAEC-PAD Supplemental Table S2) Rha and GalA the building 276
blocks of the RG I backbone represent around 90 of total mucilage extracted from wild-type 277
Arabidopsis seeds (Fig 4 Voiniciuc et al 2015c) The muci70-1 and muci70-2 mutations 278
reduced the absolute levels of Rha and GalA in total mucilage extracts by more than 50 279
compared to the wild-type control (Fig 4B) The gaut11-3 single mutant reduced pectin content 280
by around 30 compared to the wild type (Fig 4B) similar to the gaut11-4 allele (Supplemental 281
Table S2) Interestingly the absolute abundance of the minor sugars in the total mucilage 282
extracts increased by more than 40 in the muci70 and gaut11 mutants compared to wild type 283
(Supplemental Table S2) This suggests that both MUCI70 and GAUT11 are particularly 284
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10
important for the production and release of RG I but are not indispensable for the release of the 285
minor mucilage components For comparison a knockout insertion in the MYB5 transcription 286
factor which promotes seed coat differentiation and mucilage production (Li et al 2009 287
Voiniciuc et al 2015c) significantly decreased the content of all sugars found in total mucilage 288
extracts (Supplemental Table S2) Therefore muci70 and gaut11 mutants are deficient in the 289
production and release of pectic polysaccharides In contrast to the gaut11-3 and gaut11-4 290
mutants the gaut10-1 and gaut14-1 knockout mutants identified by genotyping (Supplemental 291
Table S3) reduced Rha and GalA levels by only 8 to 13 (Supplemental Table S2) Consistent 292
with their normal RR staining phenotypes (Supplemental Fig S2) gaut10-1 and gaut14-1 thus 293
had a relatively minor influence on mucilage production Indeed analysis of the muci70-1 294
gaut11-3 double revealed that MUCI70 together with GAUT11 accounted for the biosynthesis 295
and release of 88 of GalA-containing polymers in total seed mucilage extracts (Fig 4B) Two-296
factor ANOVA analysis of the HPAEC-PAD data (Supplemental Table S4) indicated that the 297
muci70-1 and gaut11-3 mutations had purely additive effects on GalA abundance Since the 298
muci70-1 gaut11-3 total mucilage extracts also contained 84 less Rha than the wild type the 299
mutated genes controlled the content of mucilage pectin in a non-redundant manner (Fig 4B) 300
Compared to the single mutants the muci70-1 gaut11-3 double mutant released even more 301
minor sugars in total mucilage extracts (Supplemental Table S2) Since the minor sugars are 302
primarily derived from hemicelluloses (Voiniciuc et al 2015a Voiniciuc et al 2015b) the 303
observed chemotype is consistent with the specific loss of pectin 304
Besides the drastic deficiency of RG I backbone sugars mutations in MUCI70 and GAUT11 305
significantly increased the absolute amounts of Gal Glc and Man in total mucilage extracts (Fig 306
4B Supplemental Table S2) but had distinct effects on the content of Ara and Xyl Based on 307
ANOVA the muci70-1 and gaut11-3 mutations had purely additive effects on the content of Gal 308
while the increases in Glc and Man content were higher than expected (Supplemental Table 309
S4) The two muci70 alleles significantly decreased Ara content (26 to 32) relative to the wild 310
type and gaut11 mutants ANOVA confirmed that only MUCI70 influenced the presence of Ara 311
(Supplemental Table S4) Surprisingly muci70 and gaut11 single mutants had polarizing effects 312
on Xyl content Relative to wild type muci70 single mutants increased Xyl abundance by 73 to 313
87 while the gaut11 single mutants and the muci70-1 gaut11-3 double mutant decreased Xyl 314
content by 43 to 47 (Supplemental Table S2) 315
To further investigate the structure of pectin and other polysaccharides glycosyl linkage 316
analysis was performed on total mucilage extracts (Table I) Relative to the wild-type control the 317
total mucilage extracts of both the muci70-1 and gaut11-3 mutants contained significant 318
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11
reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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12
sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
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693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
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- Figure 7
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- Parsed Citations
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2
Identification of Key Enzymes for Pectin Synthesis in Seed Mucilage 14
Cătălin Voiniciuc 1 Kristen A Engle1 Markus Guumlnl Sabine Dieluweit Maximilian Heinrich-15
Wilhelm Schmidt Jeong-Yeh Yang Kelley W Moremen Debra Mohnen and Bjoumlrn Usadel 16
17 Institute for Bio- and Geosciences (IBG-2 Plant Sciences) Forschungszentrum Juumllich 52425 18
Juumllich Germany (CV MH-WS MG BU) Institute for Botany and Molecular Genetics 19
(IBMG) BioSC RWTH Aachen University 52074 Aachen Germany (CV MH-WS BU) 20
Department of Biochemistry and Molecular Biology (KWM DM) Department of Plant Biology 21
(KE) University of Georgia Athens Georgia USA (KAE J-YY KWMDM) Complex 22
Carbohydrate Research Center University of Georgia Athens Georgia USA (KAE J-YY 23
KWM DM) 24
Institute of Complex Systems (ICS-7) Forschungszentrum Juumllich 52425 Juumllich Germany (SD) 25
26
E-Mails catalinvoiniciuchhude (CV) kengleugaedu (KAE) mguenlfz-juelichde 27
(MG) sdieluweitfz-juelichde (SD) mschmidtfz-juelichde (MH-WS) jyyangugaedu 28
(JYY) moremenugaedu (KWM) dmohnenccrcugaedu (DM) usadelbio1rwth-29
aachende (BU) 30
31
One-Sentence Summary 32
Mutations in two glycosyltransferase-encoding genes severely impair the elongation of pectic 33
rhamnogalacturonan I resulting in hydrophobic seeds that do not release mucilage polymers 34
35
FOOTNOTES 36
Author Contributions 37
CV designed research and wrote the article with valuable input from BU MG analyzed 38
glycosyl linkages and assisted with HPAEC-PAD work SB performed SEM analysis MS 39
cloned MUCI70 in E coli KAE and J-YY designed and carried out the GAUT11 expression 40
and enzyme analysis research with advice from KWM and DM CV performed the remaining 41
experiments All authors read the article provided comments and approved the final version 42
Funding Information 43
The research was supported by the Natural Sciences and Engineering Research Council of 44
Canada (NSERC PGS-D3 to CV) Deutsche Forschungsgemeinschaft (US9813-1) and by the 45
Ministry of Innovation Science and Research of North-Rhine Westphalia within the framework 46
of the NRW Strategieprojekt BioSC (No 313323‐400‐00213 to MH-WS and BU) The 47
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
3
research was also supported by BioEnergy Science Center Grant (DE-PS02-06ER64304) and 48
the Center for Bioenergy Innovation The BioEnergy Science Center and the Center for 49
Bioenergy Innovation are US Department of Energy Bioenergy Research Centers supported 50
by the Office of Biological and Environmental Research in the Department of Energyrsquos Office of 51
Science The research was also partially funded by the Department of Energy Center Grant DE-52
SC0015662 and US National Institutes of Health grants P41GM103390 and P01GM107012 53
Generation of the CCRC series of monoclonal antibodies used in this work was supported by a 54
grant from the NSF Plant Genome Program (DBI-0421683) 55
Present Address 56
Institute for Plant Cell Biology and Biotechnology Heinrich Heine University 40225 Duumlsseldorf 57
Germany (CV) 58
Address correspondence to catalinvoiniciuchhude 59 60 1 These authors contributed equally to the experimental work 61 62 The author responsible for distribution of materials integral to the findings presented in this 63
article in accordance with the policy described in the Instructions for Authors 64
(wwwplantphysiolorg) is Cătălin Voiniciuc (catalinvoiniciuchhude) 65
ABSTRACT 66
Pectin is a vital component of the plant cell wall and provides the molecular glue that maintains 67
cell-cell adhesion among other functions As the most complex wall polysaccharide pectin is 68
composed of several covalently-linked domains such as homogalacturonan (HG) and 69
rhamnogalacturonan I (RG I) Pectin has widespread uses in the food industry and has 70
emerging biomedical applications but its synthesis remains poorly understood For instance 71
the enzymes that catalyze RG I elongation remain unknown Recently a co-expression and 72
sequence-based MUCILAGE-RELATED (MUCI) reverse genetic screen uncovered 73
hemicellulose biosynthetic enzymes in the Arabidopsis thaliana seed coat Here we use an 74
extension of this strategy to identify MUCI70 as the founding member of a glycosyltransferase 75
family essential for the accumulation of seed mucilage a gelatinous wall rich in unbranched RG 76
I Detailed biochemical and histological characterization of two muci70 mutants and two gaut11 77
mutants identified MUCI70 and GAUT11 as required for two distinct RG I domains in seed 78
mucilage We demonstrate that unlike MUCI70 GAUT11 catalyzes HG elongation in vitro and 79
is thus likely required for the synthesis of an HG region important for RG I elongation Analysis 80
of a muci70 gaut11 double mutant confirmed that MUCI70 and GAUT11 are indispensable for 81
the production and release of the bulk of mucilage RG I and for shaping the surface 82
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
4
morphology of seeds In addition we uncover relationships between pectin and hemicelluloses 83
and show that xylan is essential for the elongation of at least one RG I domain 84
85
INTRODUCTION 86
Plant cell walls are largely composed of three major classes of polysaccharides 87
cellulose hemicellulose and pectin While cellulose and hemicelluloses are largely built of 88
neutral sugars connected by β-14-linkages pectin is defined by its high content of galacturonic 89
acid (GalA) residues connected by α-14-linkages Cellulose-hemicellulose networks have been 90
thought to provide the tensile strength of the wall while pectin was mainly implicated in cell-cell 91
adhesion and determining the porosity of the wall (Cosgrove 2016) However recent evidence 92
indicates that pectin-cellulose junctions are more prevalent than previously expected and thus 93
that pectin may play additional structural roles (Wang et al 2015) Furthermore since 94
mutations in several pectin-related genes are lethal it is evident that this matrix polysaccharide 95
has vital functions in plants (Caffall et al 2009) Pectin also has widespread uses in the food 96
industry and has emerging applications in the biomedical field including use as a gelling agent 97
for targeted drug delivery and as a bioactive molecule for cancer treatment (Maxwell et al 98
2012 Munarin et al 2012) 99
Pectin is the most complex polysaccharide in the plant cell wall consisting of multiple 100
glycan domains that may exist in one or more polymers linked via their backbones (Nakamura 101
et al 2002 Atmodjo et al 2013) The backbone of the most abundant extractable pectin 102
consists exclusively of D-GalA subunits and can be unbranched (Homogalacturonan HG) 103
substituted with D-xylose (Xyl) residues (Xylogalacturonan) or decorated with a conserved set 104
of side chains (Rhamnogalacturonan II RG II) In contrast the backbone of 105
Rhamnogalacturonan I (RG I) consists of a repeating α-D-14-GalA-α-L-12-Rha disaccharide 106
The rhamnose (Rha) residues in the RG I backbone can be frequently substituted with a wide 107
variety of oligosaccharide or polysaccharide side chains Around 40 different RG I side chain 108
structures have been reported so far (Atmodjo et al 2013) including linear β-14-linked D-109
galactan and α-15-linked L-arabinan or arabinogalactans containing both galactose (Gal) and 110
arabinose (Ara) units Despite the biochemical evidence that HG and RG I are covalently linked 111
in soybean (Nakamura et al 2002) the full in vivo structure of the pectin macromolecules has 112
yet to be determined due to the difficulty of extracting them in an intact form (Atmodjo et al 113
2013) In addition a complex proteoglycan purified from Arabidopsis (Arabidopsis thaliana) 114
suspension cultures has been shown to contain covalently linked HG and RG I domains which 115
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5
are further branched with the hemicellulose xylan (Tan et al 2013) This finding suggests that 116
certain pectin domains such as RG I may have a more central role in cell wall organization than 117
previously thought 118
Based on the large number of pectin structures that have been detected in plants their 119
biosynthesis is hypothesized to require at least 67 distinct enzymes that transfer glycosyl 120
methyl or acetyl groups (Atmodjo et al 2013) However only four types of pectin biosynthetic 121
enzymes have been identified and biochemically characterized so far These include 122
glycosyltransferase (GT) proteins that belong to four different Carbohydrate-Active Enzyme 123
(CAZy httpwwwcazyorg Lombard et al 2014) families GT8 GT47-C GT77 and GT92 124
Two GT8 proteins GALACTURONOSYLTRANSFERASE1 (GAUT1) and GAUT7 form the core 125
of a GAUT1GAUT7 complex that catalyzes the elongation of the HG backbone (Sterling et al 126
2006 Atmodjo et al 2011) Additional GAUT and GAUT-LIKE (GATL) proteins from the GT8 127
family encode proven and putative HG galacturonosyltransferases (α-GalA transferases) For 128
example GAUT4 was recently shown to be an HG α-GalA transferase whose downregulation 129
results in reduced HG and RG II production (Biswal et al 2018) Although GAUT1 and GAUT7 130
are predicted to have similar protein topologies they have surprisingly distinct functions In vivo 131
the GAUT1 enzyme is cleaved into a soluble form that is retained at the site of pectin synthesis 132
via interactions with GAUT7 a Golgi membrane-bound protein anchor with no demonstrated 133
catalytic activity (Atmodjo et al 2011) Unlike GAUT4 and the GAUT1GAUT7 complex which 134
synthesize the HG backbone the other GTs known to be involved in pectin synthesis catalyze 135
the synthesis of three distinct pectin side chains the β-13-xylosyl branches of xylogalacturonan 136
(GT47-C Jensen et al 2008) the α-13-xylosyl residues in RG II (GT77 Egelund et al 2006) 137
and the β-14-galactan side chains of RG I (GT92 Liwanag et al 2012) Overall these GT 138
activities account for only a small fraction of the pectin structures found in nature In addition 139
there is increasing evidence that seemingly distinct wall polymers such as pectin and the 140
hemicellulose xylan are structurally dependent on one another (Hao and Mohnen 2014) For 141
example the loss of GAUT12 (a GT8 protein) in the irregular xylem8 (irx8) mutant leads to 142
dwarf plants that have significant reductions in both xylan and HG (Pentildea et al 2007 Persson et 143
al 2007) Therefore the production of pectin remains poorly understood on a mechanistic level 144
and most of the molecular players involved in this process remain unknown 145
Although co-expression analysis has been a successful approach to identify GTs 146
involved in cellulose and hemicellulose biosynthesis (Brown et al 2005 Persson et al 2005) it 147
previously failed to predict obvious candidates for pectin production Two potential challenges 148
are that pectin biosynthetic enzymes may lack distinctive expression profiles in most plant 149
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
tissues and that the relevant GTs are not part of classified CAZy families (Harholt et al 2010) 150
These obstacles were surpassed by the identification of novel GT-like plant proteins through 151
Golgi proteomic studies (Nikolovski et al 2012 Nikolovski et al 2014) and the establishment 152
of Arabidopsis seed mucilage as a model for dissecting pectin synthesis (Haughn and Western 153
2012) Within a narrow developmental window Arabidopsis seed coat epidermal (SCE) cells 154
produce copious amounts of RG I along with minor amounts of cellulose hemicellulose 155
arabinogalactans and HG (Voiniciuc et al 2015c) Since at least 90 of the mucilage 156
extracted from Arabidopsis seeds consists of Rha and GalA units derived from pectin the SCE 157
cells can be exploited to identify pectin-related GTs In addition structural changes in seed 158
mucilage polysaccharides can be conveniently monitored in situ with a variety of imaging 159
techniques and specific probes (Voiniciuc et al 2018) 160
Despite the great potential of this model system only two GTs have been implicated so 161
far in the synthesis of the pectin domains in mucilage A screen of 26 gaut mutant lines for 162
altered staining of seed mucilage found only one mutant (gaut11-2) that showed smaller 163
mucilage capsules and reduced uronic acid content compared to the wild type (Caffall et al 164
2009) Although the results indicated that GAUT11 might affect HG biosynthesis in SCE cells 165
the gaut11-2 phenotype was not supported by an independent knockdown gaut11-1 allele 166
(Caffall et al 2009) GATL5 another protein from the GT8 family is the only other pectin-167
related GT that has been implicated in mucilage biosynthesis A knockout T-DNA insertion in 168
GATL5 increased the molecular weight of mucilage polysaccharides without dramatically 169
altering the glycosidic linkage composition or the content of pectin epitopes bound by antibodies 170
(Kong et al 2013) Since GATL5 was proposed to simply regulate the final size of pectin 171
polymers in mucilage additional players must be required for the elongation of RG I in 172
Arabidopsis SCE cells 173
Recently a co-expression and sequence-based MUCILAGE-RELATED (MUCI) reverse 174
genetic screen identified three GTs required for the synthesis of two distinct hemicellulosic 175
polymers (xylan and galactoglucomannan) in Arabidopsis SCE cells (Voiniciuc et al 2015b 176
Voiniciuc et al 2015a) Using an extension of this strategy we now report that the biosynthesis 177
of pectin requires MUCI70 a putative GT from an unclassified CAZy family that was not known 178
to affect cell wall structure Through a detailed biochemical and histological characterization of 179
muci70 mutants and two novel gaut11 alleles we show that these two genes are required for 180
the production of two distinct RG I domains essential for seed mucilage architecture Finally the 181
analysis of a muci70 gaut11 double mutant and the demonstration that GAUT11 is an HG α-182
GalA transferase confirms that MUCI70 and GAUT11 are indispensable for the production of 183
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two RG I domains that represent the bulk of seed mucilage and shape the surface morphology 184
of seeds 185
186
RESULTS 187
MUCI70 is a Novel Pectin-Related GT Localized in the Golgi apparatus 188
To identify novel players involved in pectin production we systematically profiled the expression 189
of all 1128 Arabidopsis thaliana members of the CAZy database (Lombard et al 2014) in the 190
seed coat using ATH1 microarray data in Genevestigator (Hruz et al 2008) This strategy 191
revealed more than 50 CAZy genes that are transcribed in the seed coat when mucilage is 192
produced The majority of these genes were not identified in the initial MUCI screen (Voiniciuc 193
et al 2015b) because they are not significantly co-expressed with known mucilage genes in 194
GeneCAT (Mutwil et al 2008) GeneMANIA (Warde-Farley et al 2010) and ATTED-II 195
(Obayashi et al 2014) Amongst this collection of genes were MUCI64IRX14 (Voiniciuc et al 196
2015a) and four members of the GAUT family (Supplemental Fig S1) including GAUT11 and 197
GATL5 Interestingly we also found one gene encoding a putative GT (At1g28240) which we 198
named MUCI70 as a promising candidate for pectin production in the Arabidopsis seed coat 199
MUCI70 represents the founding member of a GT family whose roles in cell wall biology 200
remain unclear (Fig 1A) The MUCI70 protein contains a single transmembrane domain 201
(AREMEMNON Consensus TM α-helix prediction AramTmConl Schwacke et al 2003) near its 202
N-terminus and a DUF616 (PF04765) conserved domain of unknown function (Fig 1B) 203
Phylogenetic analysis of DUF616 proteins organized MUCI70 and its six Arabidopsis paralogs 204
into four clades (Fig 1A) Each of these groups contains at least one ortholog in both 205
Physcomitrella patens and Selaginella moellendorffii members of two early diverging lineages 206
of land plants (Fig 1A) In contrast TURGOR REGULATION DEFECT 1 (TOD1 AT5G46220) 207
the only other Arabidopsis protein containing a DUF616 motif did not cluster with any of these 208
clades (Fig 1A) and appeared to be functionally distinct Indeed TOD1 was demonstrated to 209
have alkaline ceramidase activity in vitro (Chen et al 2015) rather than a CAZy-related 210
function At4g38500 a close paralog of MUCI70 (Fig 1A) was previously identified in a Golgi 211
proteomics study and showed little similarity in primary sequence and predicted 3D structure to 212
the GT8 family in Arabidopsis (Nikolovski et al 2012) Based on tight co-expression with GAUT 213
genes At4g38500 was hypothesized to be involved in pectin biosynthesis (Voxeur et al 2012) 214
MUCI70 and GAUT11 a gene that was implicated in mucilage HG biosynthesis (Caffall et 215
al 2009) showed similar transcriptional profiles in developing seeds (Supplemental Fig S1 216
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Belmonte et al 2013) and encode proteins with similar topologies (Fig 1B) GAUT11 was 217
previously found in the Golgi proteome (Parsons et al 2012) but the subcellular localization of 218
MUCI70 remained unknown To address this MUCI70 tagged with super yellow fluorescent 219
protein (sYFP) was stably expressed in Arabidopsis using the constitutive 35S promoter 220
MUCI70-sYFP was observed in intracellular punctae (Fig 1C) that co-localized with the Golgi 221
marker sialyltransferase (ST) tagged with red fluorescent protein (ST-RFP Figs 1D and 1E) 222
which marks the site of pectin production in plants 223
224
Mutations in MUCI70 and GAUT11 Cause Severe Mucilage Defects 225
To investigate the biological role of MUCI70 in SCE cells we obtained two independent T-226
DNA lines and identified homozygous mutants (Fig 2A Supplemental Table S1) While GATL5 227
was unambiguously shown to be required for mucilage pectin structure (Kong et al 2013) only 228
one of two transcriptional knockdown mutants (gaut11-2 Fig 2A) previously indicated that 229
GAUT11 influences mucilage structure (Caffall et al 2009) Therefore we analyzed two muci70 230
insertional mutants alongside two gaut11 mutants gaut11-3 and gaut11-4 with insertions in 231
GAUT11 exons (Fig 2A) Using reverse transcription quantitative polymerase chain reaction 232
(RT-qPCR) we validated that both MUCI70 and GAUT11 were transcribed in developing 233
Arabidopsis siliques from 3 to 10 days post anthesis (DPA) GAUT11 showed a dramatic 234
increase (around 15-fold) in expression at 7 DPA when pectin synthesis in SCE cells is at its 235
peak (Fig 2B) Based on the results of public microarray datasets (Winter et al 2007 Belmonte 236
et al 2013) both genes were preferentially expressed in the seed coat relative to the embryo 237
(Fig 2C) and had similar transcript levels from the heart stage (~3 DPA) to the mature green 238
stage (~10 DPA) Each insertion in the MUCI70 gene reduced its expression by at least 60 239
(Fig 2D) Although gaut11-3 and gaut11-4 did not significantly alter GAUT11 transcription at 240
either the 5 or 3 end (Fig 2D) these alleles and the previously described gaut11-2 (Caffall et 241
al 2009) are exonic insertions (Fig 2A) that likely disrupt the GAUT11 protein sequence 242
In contrast to wild-type seeds which are surrounded by large mucilage capsules (Fig 3A) 243
two muci70 and two gaut11 homozygous mutants showed severe ruthenium red (RR) staining 244
defects (Fig 3B to 3E) consisting of patchy or completely impaired mucilage release 245
Consequently the muci70-1 muci70-2 and gaut11-3 seeds were surrounded by significantly 246
smaller mucilage capsules (Fig 2E) whose surface area was only 19 to 39 of the wild-type 247
value At least 65 of muci70 and gaut11 seeds floated on water (Fig 2F Fig 3 marked by 248
stars) whereas wild-type seeds did not float (Fig 2F) despite having similar dimensions (Fig 249
2E) Besides GAUT11 three other GAUT genes (GAUT8 GAUT10 GAUT14) were expressed 250
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in the developing seed coat (Supplemental Fig S1) The gaut8 mutant was previously found to 251
be lethal but the gaut10-1 and gaut14-1 transcriptional knockout mutants were viable (Caffall et 252
al 2009) and re-examined in this study In contrast to muci70 and gaut11 mutants gaut10-1 253
and gaut14-1 did not disrupt the dimensions of the seeds or the surrounding RR-stained 254
mucilage capsules (Fig 2E Supplemental Fig S2) Therefore only one of the GAUT genes 255
tested was essential on its own for maintaining mucilage architecture consistent with the 256
previous study of the whole GAUT family (Caffall et al 2009) 257
Since both gaut11-3 and gaut11-4 mutants showed similar mucilage staining defects to the 258
previously described gaut11-2 allele (Caffall et al 2009) we primarily used gaut11-3 for further 259
experiments To investigate if MUCI70 and GAUT11 function in the same pathway we crossed 260
the muci70-1 and gaut11-3 single mutants While muci70 and gaut11 single mutants showed 261
smaller RR-stained mucilage capsules than the wild type (Fig 3A to 3E) all muci70 gaut11 262
double mutant seeds failed to release mucilage (Fig 3F) and thus floated on water (Fig 2F) 263
Despite the severe mucilage defects the muci70 gaut11 seeds were only 6 smaller than the 264
wild type (Fig 2E) This suggested that both MUCI70 and GAUT11 might be required for the 265
biosynthesis of pectin in SCE cells which is ultimately released as a hydrophilic capsule from 266
mature seeds 267
268
MUCI70 and GAUT11 Are Important for Pectin Production in SCE Cells 269
To identify the underlying biochemical defects that lead to impaired mucilage release total 270
mucilage was extracted from seeds vigorously mixed using a ball mill (Voiniciuc et al 2015b 271
Voiniciuc and Guumlnl 2016) As previously described this intensive mechanical agitation 272
effectively removes all mucilage polysaccharides resulting in seeds that are no longer stained 273
by RR (Fig 4A) The monosaccharide composition of the total mucilage extracted from hydrated 274
seeds was quantified using high-performance anion-exchange chromatography with pulsed 275
amperometric detection (HPAEC-PAD Supplemental Table S2) Rha and GalA the building 276
blocks of the RG I backbone represent around 90 of total mucilage extracted from wild-type 277
Arabidopsis seeds (Fig 4 Voiniciuc et al 2015c) The muci70-1 and muci70-2 mutations 278
reduced the absolute levels of Rha and GalA in total mucilage extracts by more than 50 279
compared to the wild-type control (Fig 4B) The gaut11-3 single mutant reduced pectin content 280
by around 30 compared to the wild type (Fig 4B) similar to the gaut11-4 allele (Supplemental 281
Table S2) Interestingly the absolute abundance of the minor sugars in the total mucilage 282
extracts increased by more than 40 in the muci70 and gaut11 mutants compared to wild type 283
(Supplemental Table S2) This suggests that both MUCI70 and GAUT11 are particularly 284
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important for the production and release of RG I but are not indispensable for the release of the 285
minor mucilage components For comparison a knockout insertion in the MYB5 transcription 286
factor which promotes seed coat differentiation and mucilage production (Li et al 2009 287
Voiniciuc et al 2015c) significantly decreased the content of all sugars found in total mucilage 288
extracts (Supplemental Table S2) Therefore muci70 and gaut11 mutants are deficient in the 289
production and release of pectic polysaccharides In contrast to the gaut11-3 and gaut11-4 290
mutants the gaut10-1 and gaut14-1 knockout mutants identified by genotyping (Supplemental 291
Table S3) reduced Rha and GalA levels by only 8 to 13 (Supplemental Table S2) Consistent 292
with their normal RR staining phenotypes (Supplemental Fig S2) gaut10-1 and gaut14-1 thus 293
had a relatively minor influence on mucilage production Indeed analysis of the muci70-1 294
gaut11-3 double revealed that MUCI70 together with GAUT11 accounted for the biosynthesis 295
and release of 88 of GalA-containing polymers in total seed mucilage extracts (Fig 4B) Two-296
factor ANOVA analysis of the HPAEC-PAD data (Supplemental Table S4) indicated that the 297
muci70-1 and gaut11-3 mutations had purely additive effects on GalA abundance Since the 298
muci70-1 gaut11-3 total mucilage extracts also contained 84 less Rha than the wild type the 299
mutated genes controlled the content of mucilage pectin in a non-redundant manner (Fig 4B) 300
Compared to the single mutants the muci70-1 gaut11-3 double mutant released even more 301
minor sugars in total mucilage extracts (Supplemental Table S2) Since the minor sugars are 302
primarily derived from hemicelluloses (Voiniciuc et al 2015a Voiniciuc et al 2015b) the 303
observed chemotype is consistent with the specific loss of pectin 304
Besides the drastic deficiency of RG I backbone sugars mutations in MUCI70 and GAUT11 305
significantly increased the absolute amounts of Gal Glc and Man in total mucilage extracts (Fig 306
4B Supplemental Table S2) but had distinct effects on the content of Ara and Xyl Based on 307
ANOVA the muci70-1 and gaut11-3 mutations had purely additive effects on the content of Gal 308
while the increases in Glc and Man content were higher than expected (Supplemental Table 309
S4) The two muci70 alleles significantly decreased Ara content (26 to 32) relative to the wild 310
type and gaut11 mutants ANOVA confirmed that only MUCI70 influenced the presence of Ara 311
(Supplemental Table S4) Surprisingly muci70 and gaut11 single mutants had polarizing effects 312
on Xyl content Relative to wild type muci70 single mutants increased Xyl abundance by 73 to 313
87 while the gaut11 single mutants and the muci70-1 gaut11-3 double mutant decreased Xyl 314
content by 43 to 47 (Supplemental Table S2) 315
To further investigate the structure of pectin and other polysaccharides glycosyl linkage 316
analysis was performed on total mucilage extracts (Table I) Relative to the wild-type control the 317
total mucilage extracts of both the muci70-1 and gaut11-3 mutants contained significant 318
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reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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18
accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
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22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
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23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
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24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
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25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
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26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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- Parsed Citations
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research was also supported by BioEnergy Science Center Grant (DE-PS02-06ER64304) and 48
the Center for Bioenergy Innovation The BioEnergy Science Center and the Center for 49
Bioenergy Innovation are US Department of Energy Bioenergy Research Centers supported 50
by the Office of Biological and Environmental Research in the Department of Energyrsquos Office of 51
Science The research was also partially funded by the Department of Energy Center Grant DE-52
SC0015662 and US National Institutes of Health grants P41GM103390 and P01GM107012 53
Generation of the CCRC series of monoclonal antibodies used in this work was supported by a 54
grant from the NSF Plant Genome Program (DBI-0421683) 55
Present Address 56
Institute for Plant Cell Biology and Biotechnology Heinrich Heine University 40225 Duumlsseldorf 57
Germany (CV) 58
Address correspondence to catalinvoiniciuchhude 59 60 1 These authors contributed equally to the experimental work 61 62 The author responsible for distribution of materials integral to the findings presented in this 63
article in accordance with the policy described in the Instructions for Authors 64
(wwwplantphysiolorg) is Cătălin Voiniciuc (catalinvoiniciuchhude) 65
ABSTRACT 66
Pectin is a vital component of the plant cell wall and provides the molecular glue that maintains 67
cell-cell adhesion among other functions As the most complex wall polysaccharide pectin is 68
composed of several covalently-linked domains such as homogalacturonan (HG) and 69
rhamnogalacturonan I (RG I) Pectin has widespread uses in the food industry and has 70
emerging biomedical applications but its synthesis remains poorly understood For instance 71
the enzymes that catalyze RG I elongation remain unknown Recently a co-expression and 72
sequence-based MUCILAGE-RELATED (MUCI) reverse genetic screen uncovered 73
hemicellulose biosynthetic enzymes in the Arabidopsis thaliana seed coat Here we use an 74
extension of this strategy to identify MUCI70 as the founding member of a glycosyltransferase 75
family essential for the accumulation of seed mucilage a gelatinous wall rich in unbranched RG 76
I Detailed biochemical and histological characterization of two muci70 mutants and two gaut11 77
mutants identified MUCI70 and GAUT11 as required for two distinct RG I domains in seed 78
mucilage We demonstrate that unlike MUCI70 GAUT11 catalyzes HG elongation in vitro and 79
is thus likely required for the synthesis of an HG region important for RG I elongation Analysis 80
of a muci70 gaut11 double mutant confirmed that MUCI70 and GAUT11 are indispensable for 81
the production and release of the bulk of mucilage RG I and for shaping the surface 82
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
4
morphology of seeds In addition we uncover relationships between pectin and hemicelluloses 83
and show that xylan is essential for the elongation of at least one RG I domain 84
85
INTRODUCTION 86
Plant cell walls are largely composed of three major classes of polysaccharides 87
cellulose hemicellulose and pectin While cellulose and hemicelluloses are largely built of 88
neutral sugars connected by β-14-linkages pectin is defined by its high content of galacturonic 89
acid (GalA) residues connected by α-14-linkages Cellulose-hemicellulose networks have been 90
thought to provide the tensile strength of the wall while pectin was mainly implicated in cell-cell 91
adhesion and determining the porosity of the wall (Cosgrove 2016) However recent evidence 92
indicates that pectin-cellulose junctions are more prevalent than previously expected and thus 93
that pectin may play additional structural roles (Wang et al 2015) Furthermore since 94
mutations in several pectin-related genes are lethal it is evident that this matrix polysaccharide 95
has vital functions in plants (Caffall et al 2009) Pectin also has widespread uses in the food 96
industry and has emerging applications in the biomedical field including use as a gelling agent 97
for targeted drug delivery and as a bioactive molecule for cancer treatment (Maxwell et al 98
2012 Munarin et al 2012) 99
Pectin is the most complex polysaccharide in the plant cell wall consisting of multiple 100
glycan domains that may exist in one or more polymers linked via their backbones (Nakamura 101
et al 2002 Atmodjo et al 2013) The backbone of the most abundant extractable pectin 102
consists exclusively of D-GalA subunits and can be unbranched (Homogalacturonan HG) 103
substituted with D-xylose (Xyl) residues (Xylogalacturonan) or decorated with a conserved set 104
of side chains (Rhamnogalacturonan II RG II) In contrast the backbone of 105
Rhamnogalacturonan I (RG I) consists of a repeating α-D-14-GalA-α-L-12-Rha disaccharide 106
The rhamnose (Rha) residues in the RG I backbone can be frequently substituted with a wide 107
variety of oligosaccharide or polysaccharide side chains Around 40 different RG I side chain 108
structures have been reported so far (Atmodjo et al 2013) including linear β-14-linked D-109
galactan and α-15-linked L-arabinan or arabinogalactans containing both galactose (Gal) and 110
arabinose (Ara) units Despite the biochemical evidence that HG and RG I are covalently linked 111
in soybean (Nakamura et al 2002) the full in vivo structure of the pectin macromolecules has 112
yet to be determined due to the difficulty of extracting them in an intact form (Atmodjo et al 113
2013) In addition a complex proteoglycan purified from Arabidopsis (Arabidopsis thaliana) 114
suspension cultures has been shown to contain covalently linked HG and RG I domains which 115
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5
are further branched with the hemicellulose xylan (Tan et al 2013) This finding suggests that 116
certain pectin domains such as RG I may have a more central role in cell wall organization than 117
previously thought 118
Based on the large number of pectin structures that have been detected in plants their 119
biosynthesis is hypothesized to require at least 67 distinct enzymes that transfer glycosyl 120
methyl or acetyl groups (Atmodjo et al 2013) However only four types of pectin biosynthetic 121
enzymes have been identified and biochemically characterized so far These include 122
glycosyltransferase (GT) proteins that belong to four different Carbohydrate-Active Enzyme 123
(CAZy httpwwwcazyorg Lombard et al 2014) families GT8 GT47-C GT77 and GT92 124
Two GT8 proteins GALACTURONOSYLTRANSFERASE1 (GAUT1) and GAUT7 form the core 125
of a GAUT1GAUT7 complex that catalyzes the elongation of the HG backbone (Sterling et al 126
2006 Atmodjo et al 2011) Additional GAUT and GAUT-LIKE (GATL) proteins from the GT8 127
family encode proven and putative HG galacturonosyltransferases (α-GalA transferases) For 128
example GAUT4 was recently shown to be an HG α-GalA transferase whose downregulation 129
results in reduced HG and RG II production (Biswal et al 2018) Although GAUT1 and GAUT7 130
are predicted to have similar protein topologies they have surprisingly distinct functions In vivo 131
the GAUT1 enzyme is cleaved into a soluble form that is retained at the site of pectin synthesis 132
via interactions with GAUT7 a Golgi membrane-bound protein anchor with no demonstrated 133
catalytic activity (Atmodjo et al 2011) Unlike GAUT4 and the GAUT1GAUT7 complex which 134
synthesize the HG backbone the other GTs known to be involved in pectin synthesis catalyze 135
the synthesis of three distinct pectin side chains the β-13-xylosyl branches of xylogalacturonan 136
(GT47-C Jensen et al 2008) the α-13-xylosyl residues in RG II (GT77 Egelund et al 2006) 137
and the β-14-galactan side chains of RG I (GT92 Liwanag et al 2012) Overall these GT 138
activities account for only a small fraction of the pectin structures found in nature In addition 139
there is increasing evidence that seemingly distinct wall polymers such as pectin and the 140
hemicellulose xylan are structurally dependent on one another (Hao and Mohnen 2014) For 141
example the loss of GAUT12 (a GT8 protein) in the irregular xylem8 (irx8) mutant leads to 142
dwarf plants that have significant reductions in both xylan and HG (Pentildea et al 2007 Persson et 143
al 2007) Therefore the production of pectin remains poorly understood on a mechanistic level 144
and most of the molecular players involved in this process remain unknown 145
Although co-expression analysis has been a successful approach to identify GTs 146
involved in cellulose and hemicellulose biosynthesis (Brown et al 2005 Persson et al 2005) it 147
previously failed to predict obvious candidates for pectin production Two potential challenges 148
are that pectin biosynthetic enzymes may lack distinctive expression profiles in most plant 149
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
tissues and that the relevant GTs are not part of classified CAZy families (Harholt et al 2010) 150
These obstacles were surpassed by the identification of novel GT-like plant proteins through 151
Golgi proteomic studies (Nikolovski et al 2012 Nikolovski et al 2014) and the establishment 152
of Arabidopsis seed mucilage as a model for dissecting pectin synthesis (Haughn and Western 153
2012) Within a narrow developmental window Arabidopsis seed coat epidermal (SCE) cells 154
produce copious amounts of RG I along with minor amounts of cellulose hemicellulose 155
arabinogalactans and HG (Voiniciuc et al 2015c) Since at least 90 of the mucilage 156
extracted from Arabidopsis seeds consists of Rha and GalA units derived from pectin the SCE 157
cells can be exploited to identify pectin-related GTs In addition structural changes in seed 158
mucilage polysaccharides can be conveniently monitored in situ with a variety of imaging 159
techniques and specific probes (Voiniciuc et al 2018) 160
Despite the great potential of this model system only two GTs have been implicated so 161
far in the synthesis of the pectin domains in mucilage A screen of 26 gaut mutant lines for 162
altered staining of seed mucilage found only one mutant (gaut11-2) that showed smaller 163
mucilage capsules and reduced uronic acid content compared to the wild type (Caffall et al 164
2009) Although the results indicated that GAUT11 might affect HG biosynthesis in SCE cells 165
the gaut11-2 phenotype was not supported by an independent knockdown gaut11-1 allele 166
(Caffall et al 2009) GATL5 another protein from the GT8 family is the only other pectin-167
related GT that has been implicated in mucilage biosynthesis A knockout T-DNA insertion in 168
GATL5 increased the molecular weight of mucilage polysaccharides without dramatically 169
altering the glycosidic linkage composition or the content of pectin epitopes bound by antibodies 170
(Kong et al 2013) Since GATL5 was proposed to simply regulate the final size of pectin 171
polymers in mucilage additional players must be required for the elongation of RG I in 172
Arabidopsis SCE cells 173
Recently a co-expression and sequence-based MUCILAGE-RELATED (MUCI) reverse 174
genetic screen identified three GTs required for the synthesis of two distinct hemicellulosic 175
polymers (xylan and galactoglucomannan) in Arabidopsis SCE cells (Voiniciuc et al 2015b 176
Voiniciuc et al 2015a) Using an extension of this strategy we now report that the biosynthesis 177
of pectin requires MUCI70 a putative GT from an unclassified CAZy family that was not known 178
to affect cell wall structure Through a detailed biochemical and histological characterization of 179
muci70 mutants and two novel gaut11 alleles we show that these two genes are required for 180
the production of two distinct RG I domains essential for seed mucilage architecture Finally the 181
analysis of a muci70 gaut11 double mutant and the demonstration that GAUT11 is an HG α-182
GalA transferase confirms that MUCI70 and GAUT11 are indispensable for the production of 183
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
two RG I domains that represent the bulk of seed mucilage and shape the surface morphology 184
of seeds 185
186
RESULTS 187
MUCI70 is a Novel Pectin-Related GT Localized in the Golgi apparatus 188
To identify novel players involved in pectin production we systematically profiled the expression 189
of all 1128 Arabidopsis thaliana members of the CAZy database (Lombard et al 2014) in the 190
seed coat using ATH1 microarray data in Genevestigator (Hruz et al 2008) This strategy 191
revealed more than 50 CAZy genes that are transcribed in the seed coat when mucilage is 192
produced The majority of these genes were not identified in the initial MUCI screen (Voiniciuc 193
et al 2015b) because they are not significantly co-expressed with known mucilage genes in 194
GeneCAT (Mutwil et al 2008) GeneMANIA (Warde-Farley et al 2010) and ATTED-II 195
(Obayashi et al 2014) Amongst this collection of genes were MUCI64IRX14 (Voiniciuc et al 196
2015a) and four members of the GAUT family (Supplemental Fig S1) including GAUT11 and 197
GATL5 Interestingly we also found one gene encoding a putative GT (At1g28240) which we 198
named MUCI70 as a promising candidate for pectin production in the Arabidopsis seed coat 199
MUCI70 represents the founding member of a GT family whose roles in cell wall biology 200
remain unclear (Fig 1A) The MUCI70 protein contains a single transmembrane domain 201
(AREMEMNON Consensus TM α-helix prediction AramTmConl Schwacke et al 2003) near its 202
N-terminus and a DUF616 (PF04765) conserved domain of unknown function (Fig 1B) 203
Phylogenetic analysis of DUF616 proteins organized MUCI70 and its six Arabidopsis paralogs 204
into four clades (Fig 1A) Each of these groups contains at least one ortholog in both 205
Physcomitrella patens and Selaginella moellendorffii members of two early diverging lineages 206
of land plants (Fig 1A) In contrast TURGOR REGULATION DEFECT 1 (TOD1 AT5G46220) 207
the only other Arabidopsis protein containing a DUF616 motif did not cluster with any of these 208
clades (Fig 1A) and appeared to be functionally distinct Indeed TOD1 was demonstrated to 209
have alkaline ceramidase activity in vitro (Chen et al 2015) rather than a CAZy-related 210
function At4g38500 a close paralog of MUCI70 (Fig 1A) was previously identified in a Golgi 211
proteomics study and showed little similarity in primary sequence and predicted 3D structure to 212
the GT8 family in Arabidopsis (Nikolovski et al 2012) Based on tight co-expression with GAUT 213
genes At4g38500 was hypothesized to be involved in pectin biosynthesis (Voxeur et al 2012) 214
MUCI70 and GAUT11 a gene that was implicated in mucilage HG biosynthesis (Caffall et 215
al 2009) showed similar transcriptional profiles in developing seeds (Supplemental Fig S1 216
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8
Belmonte et al 2013) and encode proteins with similar topologies (Fig 1B) GAUT11 was 217
previously found in the Golgi proteome (Parsons et al 2012) but the subcellular localization of 218
MUCI70 remained unknown To address this MUCI70 tagged with super yellow fluorescent 219
protein (sYFP) was stably expressed in Arabidopsis using the constitutive 35S promoter 220
MUCI70-sYFP was observed in intracellular punctae (Fig 1C) that co-localized with the Golgi 221
marker sialyltransferase (ST) tagged with red fluorescent protein (ST-RFP Figs 1D and 1E) 222
which marks the site of pectin production in plants 223
224
Mutations in MUCI70 and GAUT11 Cause Severe Mucilage Defects 225
To investigate the biological role of MUCI70 in SCE cells we obtained two independent T-226
DNA lines and identified homozygous mutants (Fig 2A Supplemental Table S1) While GATL5 227
was unambiguously shown to be required for mucilage pectin structure (Kong et al 2013) only 228
one of two transcriptional knockdown mutants (gaut11-2 Fig 2A) previously indicated that 229
GAUT11 influences mucilage structure (Caffall et al 2009) Therefore we analyzed two muci70 230
insertional mutants alongside two gaut11 mutants gaut11-3 and gaut11-4 with insertions in 231
GAUT11 exons (Fig 2A) Using reverse transcription quantitative polymerase chain reaction 232
(RT-qPCR) we validated that both MUCI70 and GAUT11 were transcribed in developing 233
Arabidopsis siliques from 3 to 10 days post anthesis (DPA) GAUT11 showed a dramatic 234
increase (around 15-fold) in expression at 7 DPA when pectin synthesis in SCE cells is at its 235
peak (Fig 2B) Based on the results of public microarray datasets (Winter et al 2007 Belmonte 236
et al 2013) both genes were preferentially expressed in the seed coat relative to the embryo 237
(Fig 2C) and had similar transcript levels from the heart stage (~3 DPA) to the mature green 238
stage (~10 DPA) Each insertion in the MUCI70 gene reduced its expression by at least 60 239
(Fig 2D) Although gaut11-3 and gaut11-4 did not significantly alter GAUT11 transcription at 240
either the 5 or 3 end (Fig 2D) these alleles and the previously described gaut11-2 (Caffall et 241
al 2009) are exonic insertions (Fig 2A) that likely disrupt the GAUT11 protein sequence 242
In contrast to wild-type seeds which are surrounded by large mucilage capsules (Fig 3A) 243
two muci70 and two gaut11 homozygous mutants showed severe ruthenium red (RR) staining 244
defects (Fig 3B to 3E) consisting of patchy or completely impaired mucilage release 245
Consequently the muci70-1 muci70-2 and gaut11-3 seeds were surrounded by significantly 246
smaller mucilage capsules (Fig 2E) whose surface area was only 19 to 39 of the wild-type 247
value At least 65 of muci70 and gaut11 seeds floated on water (Fig 2F Fig 3 marked by 248
stars) whereas wild-type seeds did not float (Fig 2F) despite having similar dimensions (Fig 249
2E) Besides GAUT11 three other GAUT genes (GAUT8 GAUT10 GAUT14) were expressed 250
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9
in the developing seed coat (Supplemental Fig S1) The gaut8 mutant was previously found to 251
be lethal but the gaut10-1 and gaut14-1 transcriptional knockout mutants were viable (Caffall et 252
al 2009) and re-examined in this study In contrast to muci70 and gaut11 mutants gaut10-1 253
and gaut14-1 did not disrupt the dimensions of the seeds or the surrounding RR-stained 254
mucilage capsules (Fig 2E Supplemental Fig S2) Therefore only one of the GAUT genes 255
tested was essential on its own for maintaining mucilage architecture consistent with the 256
previous study of the whole GAUT family (Caffall et al 2009) 257
Since both gaut11-3 and gaut11-4 mutants showed similar mucilage staining defects to the 258
previously described gaut11-2 allele (Caffall et al 2009) we primarily used gaut11-3 for further 259
experiments To investigate if MUCI70 and GAUT11 function in the same pathway we crossed 260
the muci70-1 and gaut11-3 single mutants While muci70 and gaut11 single mutants showed 261
smaller RR-stained mucilage capsules than the wild type (Fig 3A to 3E) all muci70 gaut11 262
double mutant seeds failed to release mucilage (Fig 3F) and thus floated on water (Fig 2F) 263
Despite the severe mucilage defects the muci70 gaut11 seeds were only 6 smaller than the 264
wild type (Fig 2E) This suggested that both MUCI70 and GAUT11 might be required for the 265
biosynthesis of pectin in SCE cells which is ultimately released as a hydrophilic capsule from 266
mature seeds 267
268
MUCI70 and GAUT11 Are Important for Pectin Production in SCE Cells 269
To identify the underlying biochemical defects that lead to impaired mucilage release total 270
mucilage was extracted from seeds vigorously mixed using a ball mill (Voiniciuc et al 2015b 271
Voiniciuc and Guumlnl 2016) As previously described this intensive mechanical agitation 272
effectively removes all mucilage polysaccharides resulting in seeds that are no longer stained 273
by RR (Fig 4A) The monosaccharide composition of the total mucilage extracted from hydrated 274
seeds was quantified using high-performance anion-exchange chromatography with pulsed 275
amperometric detection (HPAEC-PAD Supplemental Table S2) Rha and GalA the building 276
blocks of the RG I backbone represent around 90 of total mucilage extracted from wild-type 277
Arabidopsis seeds (Fig 4 Voiniciuc et al 2015c) The muci70-1 and muci70-2 mutations 278
reduced the absolute levels of Rha and GalA in total mucilage extracts by more than 50 279
compared to the wild-type control (Fig 4B) The gaut11-3 single mutant reduced pectin content 280
by around 30 compared to the wild type (Fig 4B) similar to the gaut11-4 allele (Supplemental 281
Table S2) Interestingly the absolute abundance of the minor sugars in the total mucilage 282
extracts increased by more than 40 in the muci70 and gaut11 mutants compared to wild type 283
(Supplemental Table S2) This suggests that both MUCI70 and GAUT11 are particularly 284
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
important for the production and release of RG I but are not indispensable for the release of the 285
minor mucilage components For comparison a knockout insertion in the MYB5 transcription 286
factor which promotes seed coat differentiation and mucilage production (Li et al 2009 287
Voiniciuc et al 2015c) significantly decreased the content of all sugars found in total mucilage 288
extracts (Supplemental Table S2) Therefore muci70 and gaut11 mutants are deficient in the 289
production and release of pectic polysaccharides In contrast to the gaut11-3 and gaut11-4 290
mutants the gaut10-1 and gaut14-1 knockout mutants identified by genotyping (Supplemental 291
Table S3) reduced Rha and GalA levels by only 8 to 13 (Supplemental Table S2) Consistent 292
with their normal RR staining phenotypes (Supplemental Fig S2) gaut10-1 and gaut14-1 thus 293
had a relatively minor influence on mucilage production Indeed analysis of the muci70-1 294
gaut11-3 double revealed that MUCI70 together with GAUT11 accounted for the biosynthesis 295
and release of 88 of GalA-containing polymers in total seed mucilage extracts (Fig 4B) Two-296
factor ANOVA analysis of the HPAEC-PAD data (Supplemental Table S4) indicated that the 297
muci70-1 and gaut11-3 mutations had purely additive effects on GalA abundance Since the 298
muci70-1 gaut11-3 total mucilage extracts also contained 84 less Rha than the wild type the 299
mutated genes controlled the content of mucilage pectin in a non-redundant manner (Fig 4B) 300
Compared to the single mutants the muci70-1 gaut11-3 double mutant released even more 301
minor sugars in total mucilage extracts (Supplemental Table S2) Since the minor sugars are 302
primarily derived from hemicelluloses (Voiniciuc et al 2015a Voiniciuc et al 2015b) the 303
observed chemotype is consistent with the specific loss of pectin 304
Besides the drastic deficiency of RG I backbone sugars mutations in MUCI70 and GAUT11 305
significantly increased the absolute amounts of Gal Glc and Man in total mucilage extracts (Fig 306
4B Supplemental Table S2) but had distinct effects on the content of Ara and Xyl Based on 307
ANOVA the muci70-1 and gaut11-3 mutations had purely additive effects on the content of Gal 308
while the increases in Glc and Man content were higher than expected (Supplemental Table 309
S4) The two muci70 alleles significantly decreased Ara content (26 to 32) relative to the wild 310
type and gaut11 mutants ANOVA confirmed that only MUCI70 influenced the presence of Ara 311
(Supplemental Table S4) Surprisingly muci70 and gaut11 single mutants had polarizing effects 312
on Xyl content Relative to wild type muci70 single mutants increased Xyl abundance by 73 to 313
87 while the gaut11 single mutants and the muci70-1 gaut11-3 double mutant decreased Xyl 314
content by 43 to 47 (Supplemental Table S2) 315
To further investigate the structure of pectin and other polysaccharides glycosyl linkage 316
analysis was performed on total mucilage extracts (Table I) Relative to the wild-type control the 317
total mucilage extracts of both the muci70-1 and gaut11-3 mutants contained significant 318
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
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- Figure 8
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4
morphology of seeds In addition we uncover relationships between pectin and hemicelluloses 83
and show that xylan is essential for the elongation of at least one RG I domain 84
85
INTRODUCTION 86
Plant cell walls are largely composed of three major classes of polysaccharides 87
cellulose hemicellulose and pectin While cellulose and hemicelluloses are largely built of 88
neutral sugars connected by β-14-linkages pectin is defined by its high content of galacturonic 89
acid (GalA) residues connected by α-14-linkages Cellulose-hemicellulose networks have been 90
thought to provide the tensile strength of the wall while pectin was mainly implicated in cell-cell 91
adhesion and determining the porosity of the wall (Cosgrove 2016) However recent evidence 92
indicates that pectin-cellulose junctions are more prevalent than previously expected and thus 93
that pectin may play additional structural roles (Wang et al 2015) Furthermore since 94
mutations in several pectin-related genes are lethal it is evident that this matrix polysaccharide 95
has vital functions in plants (Caffall et al 2009) Pectin also has widespread uses in the food 96
industry and has emerging applications in the biomedical field including use as a gelling agent 97
for targeted drug delivery and as a bioactive molecule for cancer treatment (Maxwell et al 98
2012 Munarin et al 2012) 99
Pectin is the most complex polysaccharide in the plant cell wall consisting of multiple 100
glycan domains that may exist in one or more polymers linked via their backbones (Nakamura 101
et al 2002 Atmodjo et al 2013) The backbone of the most abundant extractable pectin 102
consists exclusively of D-GalA subunits and can be unbranched (Homogalacturonan HG) 103
substituted with D-xylose (Xyl) residues (Xylogalacturonan) or decorated with a conserved set 104
of side chains (Rhamnogalacturonan II RG II) In contrast the backbone of 105
Rhamnogalacturonan I (RG I) consists of a repeating α-D-14-GalA-α-L-12-Rha disaccharide 106
The rhamnose (Rha) residues in the RG I backbone can be frequently substituted with a wide 107
variety of oligosaccharide or polysaccharide side chains Around 40 different RG I side chain 108
structures have been reported so far (Atmodjo et al 2013) including linear β-14-linked D-109
galactan and α-15-linked L-arabinan or arabinogalactans containing both galactose (Gal) and 110
arabinose (Ara) units Despite the biochemical evidence that HG and RG I are covalently linked 111
in soybean (Nakamura et al 2002) the full in vivo structure of the pectin macromolecules has 112
yet to be determined due to the difficulty of extracting them in an intact form (Atmodjo et al 113
2013) In addition a complex proteoglycan purified from Arabidopsis (Arabidopsis thaliana) 114
suspension cultures has been shown to contain covalently linked HG and RG I domains which 115
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5
are further branched with the hemicellulose xylan (Tan et al 2013) This finding suggests that 116
certain pectin domains such as RG I may have a more central role in cell wall organization than 117
previously thought 118
Based on the large number of pectin structures that have been detected in plants their 119
biosynthesis is hypothesized to require at least 67 distinct enzymes that transfer glycosyl 120
methyl or acetyl groups (Atmodjo et al 2013) However only four types of pectin biosynthetic 121
enzymes have been identified and biochemically characterized so far These include 122
glycosyltransferase (GT) proteins that belong to four different Carbohydrate-Active Enzyme 123
(CAZy httpwwwcazyorg Lombard et al 2014) families GT8 GT47-C GT77 and GT92 124
Two GT8 proteins GALACTURONOSYLTRANSFERASE1 (GAUT1) and GAUT7 form the core 125
of a GAUT1GAUT7 complex that catalyzes the elongation of the HG backbone (Sterling et al 126
2006 Atmodjo et al 2011) Additional GAUT and GAUT-LIKE (GATL) proteins from the GT8 127
family encode proven and putative HG galacturonosyltransferases (α-GalA transferases) For 128
example GAUT4 was recently shown to be an HG α-GalA transferase whose downregulation 129
results in reduced HG and RG II production (Biswal et al 2018) Although GAUT1 and GAUT7 130
are predicted to have similar protein topologies they have surprisingly distinct functions In vivo 131
the GAUT1 enzyme is cleaved into a soluble form that is retained at the site of pectin synthesis 132
via interactions with GAUT7 a Golgi membrane-bound protein anchor with no demonstrated 133
catalytic activity (Atmodjo et al 2011) Unlike GAUT4 and the GAUT1GAUT7 complex which 134
synthesize the HG backbone the other GTs known to be involved in pectin synthesis catalyze 135
the synthesis of three distinct pectin side chains the β-13-xylosyl branches of xylogalacturonan 136
(GT47-C Jensen et al 2008) the α-13-xylosyl residues in RG II (GT77 Egelund et al 2006) 137
and the β-14-galactan side chains of RG I (GT92 Liwanag et al 2012) Overall these GT 138
activities account for only a small fraction of the pectin structures found in nature In addition 139
there is increasing evidence that seemingly distinct wall polymers such as pectin and the 140
hemicellulose xylan are structurally dependent on one another (Hao and Mohnen 2014) For 141
example the loss of GAUT12 (a GT8 protein) in the irregular xylem8 (irx8) mutant leads to 142
dwarf plants that have significant reductions in both xylan and HG (Pentildea et al 2007 Persson et 143
al 2007) Therefore the production of pectin remains poorly understood on a mechanistic level 144
and most of the molecular players involved in this process remain unknown 145
Although co-expression analysis has been a successful approach to identify GTs 146
involved in cellulose and hemicellulose biosynthesis (Brown et al 2005 Persson et al 2005) it 147
previously failed to predict obvious candidates for pectin production Two potential challenges 148
are that pectin biosynthetic enzymes may lack distinctive expression profiles in most plant 149
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
tissues and that the relevant GTs are not part of classified CAZy families (Harholt et al 2010) 150
These obstacles were surpassed by the identification of novel GT-like plant proteins through 151
Golgi proteomic studies (Nikolovski et al 2012 Nikolovski et al 2014) and the establishment 152
of Arabidopsis seed mucilage as a model for dissecting pectin synthesis (Haughn and Western 153
2012) Within a narrow developmental window Arabidopsis seed coat epidermal (SCE) cells 154
produce copious amounts of RG I along with minor amounts of cellulose hemicellulose 155
arabinogalactans and HG (Voiniciuc et al 2015c) Since at least 90 of the mucilage 156
extracted from Arabidopsis seeds consists of Rha and GalA units derived from pectin the SCE 157
cells can be exploited to identify pectin-related GTs In addition structural changes in seed 158
mucilage polysaccharides can be conveniently monitored in situ with a variety of imaging 159
techniques and specific probes (Voiniciuc et al 2018) 160
Despite the great potential of this model system only two GTs have been implicated so 161
far in the synthesis of the pectin domains in mucilage A screen of 26 gaut mutant lines for 162
altered staining of seed mucilage found only one mutant (gaut11-2) that showed smaller 163
mucilage capsules and reduced uronic acid content compared to the wild type (Caffall et al 164
2009) Although the results indicated that GAUT11 might affect HG biosynthesis in SCE cells 165
the gaut11-2 phenotype was not supported by an independent knockdown gaut11-1 allele 166
(Caffall et al 2009) GATL5 another protein from the GT8 family is the only other pectin-167
related GT that has been implicated in mucilage biosynthesis A knockout T-DNA insertion in 168
GATL5 increased the molecular weight of mucilage polysaccharides without dramatically 169
altering the glycosidic linkage composition or the content of pectin epitopes bound by antibodies 170
(Kong et al 2013) Since GATL5 was proposed to simply regulate the final size of pectin 171
polymers in mucilage additional players must be required for the elongation of RG I in 172
Arabidopsis SCE cells 173
Recently a co-expression and sequence-based MUCILAGE-RELATED (MUCI) reverse 174
genetic screen identified three GTs required for the synthesis of two distinct hemicellulosic 175
polymers (xylan and galactoglucomannan) in Arabidopsis SCE cells (Voiniciuc et al 2015b 176
Voiniciuc et al 2015a) Using an extension of this strategy we now report that the biosynthesis 177
of pectin requires MUCI70 a putative GT from an unclassified CAZy family that was not known 178
to affect cell wall structure Through a detailed biochemical and histological characterization of 179
muci70 mutants and two novel gaut11 alleles we show that these two genes are required for 180
the production of two distinct RG I domains essential for seed mucilage architecture Finally the 181
analysis of a muci70 gaut11 double mutant and the demonstration that GAUT11 is an HG α-182
GalA transferase confirms that MUCI70 and GAUT11 are indispensable for the production of 183
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7
two RG I domains that represent the bulk of seed mucilage and shape the surface morphology 184
of seeds 185
186
RESULTS 187
MUCI70 is a Novel Pectin-Related GT Localized in the Golgi apparatus 188
To identify novel players involved in pectin production we systematically profiled the expression 189
of all 1128 Arabidopsis thaliana members of the CAZy database (Lombard et al 2014) in the 190
seed coat using ATH1 microarray data in Genevestigator (Hruz et al 2008) This strategy 191
revealed more than 50 CAZy genes that are transcribed in the seed coat when mucilage is 192
produced The majority of these genes were not identified in the initial MUCI screen (Voiniciuc 193
et al 2015b) because they are not significantly co-expressed with known mucilage genes in 194
GeneCAT (Mutwil et al 2008) GeneMANIA (Warde-Farley et al 2010) and ATTED-II 195
(Obayashi et al 2014) Amongst this collection of genes were MUCI64IRX14 (Voiniciuc et al 196
2015a) and four members of the GAUT family (Supplemental Fig S1) including GAUT11 and 197
GATL5 Interestingly we also found one gene encoding a putative GT (At1g28240) which we 198
named MUCI70 as a promising candidate for pectin production in the Arabidopsis seed coat 199
MUCI70 represents the founding member of a GT family whose roles in cell wall biology 200
remain unclear (Fig 1A) The MUCI70 protein contains a single transmembrane domain 201
(AREMEMNON Consensus TM α-helix prediction AramTmConl Schwacke et al 2003) near its 202
N-terminus and a DUF616 (PF04765) conserved domain of unknown function (Fig 1B) 203
Phylogenetic analysis of DUF616 proteins organized MUCI70 and its six Arabidopsis paralogs 204
into four clades (Fig 1A) Each of these groups contains at least one ortholog in both 205
Physcomitrella patens and Selaginella moellendorffii members of two early diverging lineages 206
of land plants (Fig 1A) In contrast TURGOR REGULATION DEFECT 1 (TOD1 AT5G46220) 207
the only other Arabidopsis protein containing a DUF616 motif did not cluster with any of these 208
clades (Fig 1A) and appeared to be functionally distinct Indeed TOD1 was demonstrated to 209
have alkaline ceramidase activity in vitro (Chen et al 2015) rather than a CAZy-related 210
function At4g38500 a close paralog of MUCI70 (Fig 1A) was previously identified in a Golgi 211
proteomics study and showed little similarity in primary sequence and predicted 3D structure to 212
the GT8 family in Arabidopsis (Nikolovski et al 2012) Based on tight co-expression with GAUT 213
genes At4g38500 was hypothesized to be involved in pectin biosynthesis (Voxeur et al 2012) 214
MUCI70 and GAUT11 a gene that was implicated in mucilage HG biosynthesis (Caffall et 215
al 2009) showed similar transcriptional profiles in developing seeds (Supplemental Fig S1 216
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Belmonte et al 2013) and encode proteins with similar topologies (Fig 1B) GAUT11 was 217
previously found in the Golgi proteome (Parsons et al 2012) but the subcellular localization of 218
MUCI70 remained unknown To address this MUCI70 tagged with super yellow fluorescent 219
protein (sYFP) was stably expressed in Arabidopsis using the constitutive 35S promoter 220
MUCI70-sYFP was observed in intracellular punctae (Fig 1C) that co-localized with the Golgi 221
marker sialyltransferase (ST) tagged with red fluorescent protein (ST-RFP Figs 1D and 1E) 222
which marks the site of pectin production in plants 223
224
Mutations in MUCI70 and GAUT11 Cause Severe Mucilage Defects 225
To investigate the biological role of MUCI70 in SCE cells we obtained two independent T-226
DNA lines and identified homozygous mutants (Fig 2A Supplemental Table S1) While GATL5 227
was unambiguously shown to be required for mucilage pectin structure (Kong et al 2013) only 228
one of two transcriptional knockdown mutants (gaut11-2 Fig 2A) previously indicated that 229
GAUT11 influences mucilage structure (Caffall et al 2009) Therefore we analyzed two muci70 230
insertional mutants alongside two gaut11 mutants gaut11-3 and gaut11-4 with insertions in 231
GAUT11 exons (Fig 2A) Using reverse transcription quantitative polymerase chain reaction 232
(RT-qPCR) we validated that both MUCI70 and GAUT11 were transcribed in developing 233
Arabidopsis siliques from 3 to 10 days post anthesis (DPA) GAUT11 showed a dramatic 234
increase (around 15-fold) in expression at 7 DPA when pectin synthesis in SCE cells is at its 235
peak (Fig 2B) Based on the results of public microarray datasets (Winter et al 2007 Belmonte 236
et al 2013) both genes were preferentially expressed in the seed coat relative to the embryo 237
(Fig 2C) and had similar transcript levels from the heart stage (~3 DPA) to the mature green 238
stage (~10 DPA) Each insertion in the MUCI70 gene reduced its expression by at least 60 239
(Fig 2D) Although gaut11-3 and gaut11-4 did not significantly alter GAUT11 transcription at 240
either the 5 or 3 end (Fig 2D) these alleles and the previously described gaut11-2 (Caffall et 241
al 2009) are exonic insertions (Fig 2A) that likely disrupt the GAUT11 protein sequence 242
In contrast to wild-type seeds which are surrounded by large mucilage capsules (Fig 3A) 243
two muci70 and two gaut11 homozygous mutants showed severe ruthenium red (RR) staining 244
defects (Fig 3B to 3E) consisting of patchy or completely impaired mucilage release 245
Consequently the muci70-1 muci70-2 and gaut11-3 seeds were surrounded by significantly 246
smaller mucilage capsules (Fig 2E) whose surface area was only 19 to 39 of the wild-type 247
value At least 65 of muci70 and gaut11 seeds floated on water (Fig 2F Fig 3 marked by 248
stars) whereas wild-type seeds did not float (Fig 2F) despite having similar dimensions (Fig 249
2E) Besides GAUT11 three other GAUT genes (GAUT8 GAUT10 GAUT14) were expressed 250
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in the developing seed coat (Supplemental Fig S1) The gaut8 mutant was previously found to 251
be lethal but the gaut10-1 and gaut14-1 transcriptional knockout mutants were viable (Caffall et 252
al 2009) and re-examined in this study In contrast to muci70 and gaut11 mutants gaut10-1 253
and gaut14-1 did not disrupt the dimensions of the seeds or the surrounding RR-stained 254
mucilage capsules (Fig 2E Supplemental Fig S2) Therefore only one of the GAUT genes 255
tested was essential on its own for maintaining mucilage architecture consistent with the 256
previous study of the whole GAUT family (Caffall et al 2009) 257
Since both gaut11-3 and gaut11-4 mutants showed similar mucilage staining defects to the 258
previously described gaut11-2 allele (Caffall et al 2009) we primarily used gaut11-3 for further 259
experiments To investigate if MUCI70 and GAUT11 function in the same pathway we crossed 260
the muci70-1 and gaut11-3 single mutants While muci70 and gaut11 single mutants showed 261
smaller RR-stained mucilage capsules than the wild type (Fig 3A to 3E) all muci70 gaut11 262
double mutant seeds failed to release mucilage (Fig 3F) and thus floated on water (Fig 2F) 263
Despite the severe mucilage defects the muci70 gaut11 seeds were only 6 smaller than the 264
wild type (Fig 2E) This suggested that both MUCI70 and GAUT11 might be required for the 265
biosynthesis of pectin in SCE cells which is ultimately released as a hydrophilic capsule from 266
mature seeds 267
268
MUCI70 and GAUT11 Are Important for Pectin Production in SCE Cells 269
To identify the underlying biochemical defects that lead to impaired mucilage release total 270
mucilage was extracted from seeds vigorously mixed using a ball mill (Voiniciuc et al 2015b 271
Voiniciuc and Guumlnl 2016) As previously described this intensive mechanical agitation 272
effectively removes all mucilage polysaccharides resulting in seeds that are no longer stained 273
by RR (Fig 4A) The monosaccharide composition of the total mucilage extracted from hydrated 274
seeds was quantified using high-performance anion-exchange chromatography with pulsed 275
amperometric detection (HPAEC-PAD Supplemental Table S2) Rha and GalA the building 276
blocks of the RG I backbone represent around 90 of total mucilage extracted from wild-type 277
Arabidopsis seeds (Fig 4 Voiniciuc et al 2015c) The muci70-1 and muci70-2 mutations 278
reduced the absolute levels of Rha and GalA in total mucilage extracts by more than 50 279
compared to the wild-type control (Fig 4B) The gaut11-3 single mutant reduced pectin content 280
by around 30 compared to the wild type (Fig 4B) similar to the gaut11-4 allele (Supplemental 281
Table S2) Interestingly the absolute abundance of the minor sugars in the total mucilage 282
extracts increased by more than 40 in the muci70 and gaut11 mutants compared to wild type 283
(Supplemental Table S2) This suggests that both MUCI70 and GAUT11 are particularly 284
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important for the production and release of RG I but are not indispensable for the release of the 285
minor mucilage components For comparison a knockout insertion in the MYB5 transcription 286
factor which promotes seed coat differentiation and mucilage production (Li et al 2009 287
Voiniciuc et al 2015c) significantly decreased the content of all sugars found in total mucilage 288
extracts (Supplemental Table S2) Therefore muci70 and gaut11 mutants are deficient in the 289
production and release of pectic polysaccharides In contrast to the gaut11-3 and gaut11-4 290
mutants the gaut10-1 and gaut14-1 knockout mutants identified by genotyping (Supplemental 291
Table S3) reduced Rha and GalA levels by only 8 to 13 (Supplemental Table S2) Consistent 292
with their normal RR staining phenotypes (Supplemental Fig S2) gaut10-1 and gaut14-1 thus 293
had a relatively minor influence on mucilage production Indeed analysis of the muci70-1 294
gaut11-3 double revealed that MUCI70 together with GAUT11 accounted for the biosynthesis 295
and release of 88 of GalA-containing polymers in total seed mucilage extracts (Fig 4B) Two-296
factor ANOVA analysis of the HPAEC-PAD data (Supplemental Table S4) indicated that the 297
muci70-1 and gaut11-3 mutations had purely additive effects on GalA abundance Since the 298
muci70-1 gaut11-3 total mucilage extracts also contained 84 less Rha than the wild type the 299
mutated genes controlled the content of mucilage pectin in a non-redundant manner (Fig 4B) 300
Compared to the single mutants the muci70-1 gaut11-3 double mutant released even more 301
minor sugars in total mucilage extracts (Supplemental Table S2) Since the minor sugars are 302
primarily derived from hemicelluloses (Voiniciuc et al 2015a Voiniciuc et al 2015b) the 303
observed chemotype is consistent with the specific loss of pectin 304
Besides the drastic deficiency of RG I backbone sugars mutations in MUCI70 and GAUT11 305
significantly increased the absolute amounts of Gal Glc and Man in total mucilage extracts (Fig 306
4B Supplemental Table S2) but had distinct effects on the content of Ara and Xyl Based on 307
ANOVA the muci70-1 and gaut11-3 mutations had purely additive effects on the content of Gal 308
while the increases in Glc and Man content were higher than expected (Supplemental Table 309
S4) The two muci70 alleles significantly decreased Ara content (26 to 32) relative to the wild 310
type and gaut11 mutants ANOVA confirmed that only MUCI70 influenced the presence of Ara 311
(Supplemental Table S4) Surprisingly muci70 and gaut11 single mutants had polarizing effects 312
on Xyl content Relative to wild type muci70 single mutants increased Xyl abundance by 73 to 313
87 while the gaut11 single mutants and the muci70-1 gaut11-3 double mutant decreased Xyl 314
content by 43 to 47 (Supplemental Table S2) 315
To further investigate the structure of pectin and other polysaccharides glycosyl linkage 316
analysis was performed on total mucilage extracts (Table I) Relative to the wild-type control the 317
total mucilage extracts of both the muci70-1 and gaut11-3 mutants contained significant 318
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reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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18
accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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19
production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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20
providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
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22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
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23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
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24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
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25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
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26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
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27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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are further branched with the hemicellulose xylan (Tan et al 2013) This finding suggests that 116
certain pectin domains such as RG I may have a more central role in cell wall organization than 117
previously thought 118
Based on the large number of pectin structures that have been detected in plants their 119
biosynthesis is hypothesized to require at least 67 distinct enzymes that transfer glycosyl 120
methyl or acetyl groups (Atmodjo et al 2013) However only four types of pectin biosynthetic 121
enzymes have been identified and biochemically characterized so far These include 122
glycosyltransferase (GT) proteins that belong to four different Carbohydrate-Active Enzyme 123
(CAZy httpwwwcazyorg Lombard et al 2014) families GT8 GT47-C GT77 and GT92 124
Two GT8 proteins GALACTURONOSYLTRANSFERASE1 (GAUT1) and GAUT7 form the core 125
of a GAUT1GAUT7 complex that catalyzes the elongation of the HG backbone (Sterling et al 126
2006 Atmodjo et al 2011) Additional GAUT and GAUT-LIKE (GATL) proteins from the GT8 127
family encode proven and putative HG galacturonosyltransferases (α-GalA transferases) For 128
example GAUT4 was recently shown to be an HG α-GalA transferase whose downregulation 129
results in reduced HG and RG II production (Biswal et al 2018) Although GAUT1 and GAUT7 130
are predicted to have similar protein topologies they have surprisingly distinct functions In vivo 131
the GAUT1 enzyme is cleaved into a soluble form that is retained at the site of pectin synthesis 132
via interactions with GAUT7 a Golgi membrane-bound protein anchor with no demonstrated 133
catalytic activity (Atmodjo et al 2011) Unlike GAUT4 and the GAUT1GAUT7 complex which 134
synthesize the HG backbone the other GTs known to be involved in pectin synthesis catalyze 135
the synthesis of three distinct pectin side chains the β-13-xylosyl branches of xylogalacturonan 136
(GT47-C Jensen et al 2008) the α-13-xylosyl residues in RG II (GT77 Egelund et al 2006) 137
and the β-14-galactan side chains of RG I (GT92 Liwanag et al 2012) Overall these GT 138
activities account for only a small fraction of the pectin structures found in nature In addition 139
there is increasing evidence that seemingly distinct wall polymers such as pectin and the 140
hemicellulose xylan are structurally dependent on one another (Hao and Mohnen 2014) For 141
example the loss of GAUT12 (a GT8 protein) in the irregular xylem8 (irx8) mutant leads to 142
dwarf plants that have significant reductions in both xylan and HG (Pentildea et al 2007 Persson et 143
al 2007) Therefore the production of pectin remains poorly understood on a mechanistic level 144
and most of the molecular players involved in this process remain unknown 145
Although co-expression analysis has been a successful approach to identify GTs 146
involved in cellulose and hemicellulose biosynthesis (Brown et al 2005 Persson et al 2005) it 147
previously failed to predict obvious candidates for pectin production Two potential challenges 148
are that pectin biosynthetic enzymes may lack distinctive expression profiles in most plant 149
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6
tissues and that the relevant GTs are not part of classified CAZy families (Harholt et al 2010) 150
These obstacles were surpassed by the identification of novel GT-like plant proteins through 151
Golgi proteomic studies (Nikolovski et al 2012 Nikolovski et al 2014) and the establishment 152
of Arabidopsis seed mucilage as a model for dissecting pectin synthesis (Haughn and Western 153
2012) Within a narrow developmental window Arabidopsis seed coat epidermal (SCE) cells 154
produce copious amounts of RG I along with minor amounts of cellulose hemicellulose 155
arabinogalactans and HG (Voiniciuc et al 2015c) Since at least 90 of the mucilage 156
extracted from Arabidopsis seeds consists of Rha and GalA units derived from pectin the SCE 157
cells can be exploited to identify pectin-related GTs In addition structural changes in seed 158
mucilage polysaccharides can be conveniently monitored in situ with a variety of imaging 159
techniques and specific probes (Voiniciuc et al 2018) 160
Despite the great potential of this model system only two GTs have been implicated so 161
far in the synthesis of the pectin domains in mucilage A screen of 26 gaut mutant lines for 162
altered staining of seed mucilage found only one mutant (gaut11-2) that showed smaller 163
mucilage capsules and reduced uronic acid content compared to the wild type (Caffall et al 164
2009) Although the results indicated that GAUT11 might affect HG biosynthesis in SCE cells 165
the gaut11-2 phenotype was not supported by an independent knockdown gaut11-1 allele 166
(Caffall et al 2009) GATL5 another protein from the GT8 family is the only other pectin-167
related GT that has been implicated in mucilage biosynthesis A knockout T-DNA insertion in 168
GATL5 increased the molecular weight of mucilage polysaccharides without dramatically 169
altering the glycosidic linkage composition or the content of pectin epitopes bound by antibodies 170
(Kong et al 2013) Since GATL5 was proposed to simply regulate the final size of pectin 171
polymers in mucilage additional players must be required for the elongation of RG I in 172
Arabidopsis SCE cells 173
Recently a co-expression and sequence-based MUCILAGE-RELATED (MUCI) reverse 174
genetic screen identified three GTs required for the synthesis of two distinct hemicellulosic 175
polymers (xylan and galactoglucomannan) in Arabidopsis SCE cells (Voiniciuc et al 2015b 176
Voiniciuc et al 2015a) Using an extension of this strategy we now report that the biosynthesis 177
of pectin requires MUCI70 a putative GT from an unclassified CAZy family that was not known 178
to affect cell wall structure Through a detailed biochemical and histological characterization of 179
muci70 mutants and two novel gaut11 alleles we show that these two genes are required for 180
the production of two distinct RG I domains essential for seed mucilage architecture Finally the 181
analysis of a muci70 gaut11 double mutant and the demonstration that GAUT11 is an HG α-182
GalA transferase confirms that MUCI70 and GAUT11 are indispensable for the production of 183
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7
two RG I domains that represent the bulk of seed mucilage and shape the surface morphology 184
of seeds 185
186
RESULTS 187
MUCI70 is a Novel Pectin-Related GT Localized in the Golgi apparatus 188
To identify novel players involved in pectin production we systematically profiled the expression 189
of all 1128 Arabidopsis thaliana members of the CAZy database (Lombard et al 2014) in the 190
seed coat using ATH1 microarray data in Genevestigator (Hruz et al 2008) This strategy 191
revealed more than 50 CAZy genes that are transcribed in the seed coat when mucilage is 192
produced The majority of these genes were not identified in the initial MUCI screen (Voiniciuc 193
et al 2015b) because they are not significantly co-expressed with known mucilage genes in 194
GeneCAT (Mutwil et al 2008) GeneMANIA (Warde-Farley et al 2010) and ATTED-II 195
(Obayashi et al 2014) Amongst this collection of genes were MUCI64IRX14 (Voiniciuc et al 196
2015a) and four members of the GAUT family (Supplemental Fig S1) including GAUT11 and 197
GATL5 Interestingly we also found one gene encoding a putative GT (At1g28240) which we 198
named MUCI70 as a promising candidate for pectin production in the Arabidopsis seed coat 199
MUCI70 represents the founding member of a GT family whose roles in cell wall biology 200
remain unclear (Fig 1A) The MUCI70 protein contains a single transmembrane domain 201
(AREMEMNON Consensus TM α-helix prediction AramTmConl Schwacke et al 2003) near its 202
N-terminus and a DUF616 (PF04765) conserved domain of unknown function (Fig 1B) 203
Phylogenetic analysis of DUF616 proteins organized MUCI70 and its six Arabidopsis paralogs 204
into four clades (Fig 1A) Each of these groups contains at least one ortholog in both 205
Physcomitrella patens and Selaginella moellendorffii members of two early diverging lineages 206
of land plants (Fig 1A) In contrast TURGOR REGULATION DEFECT 1 (TOD1 AT5G46220) 207
the only other Arabidopsis protein containing a DUF616 motif did not cluster with any of these 208
clades (Fig 1A) and appeared to be functionally distinct Indeed TOD1 was demonstrated to 209
have alkaline ceramidase activity in vitro (Chen et al 2015) rather than a CAZy-related 210
function At4g38500 a close paralog of MUCI70 (Fig 1A) was previously identified in a Golgi 211
proteomics study and showed little similarity in primary sequence and predicted 3D structure to 212
the GT8 family in Arabidopsis (Nikolovski et al 2012) Based on tight co-expression with GAUT 213
genes At4g38500 was hypothesized to be involved in pectin biosynthesis (Voxeur et al 2012) 214
MUCI70 and GAUT11 a gene that was implicated in mucilage HG biosynthesis (Caffall et 215
al 2009) showed similar transcriptional profiles in developing seeds (Supplemental Fig S1 216
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8
Belmonte et al 2013) and encode proteins with similar topologies (Fig 1B) GAUT11 was 217
previously found in the Golgi proteome (Parsons et al 2012) but the subcellular localization of 218
MUCI70 remained unknown To address this MUCI70 tagged with super yellow fluorescent 219
protein (sYFP) was stably expressed in Arabidopsis using the constitutive 35S promoter 220
MUCI70-sYFP was observed in intracellular punctae (Fig 1C) that co-localized with the Golgi 221
marker sialyltransferase (ST) tagged with red fluorescent protein (ST-RFP Figs 1D and 1E) 222
which marks the site of pectin production in plants 223
224
Mutations in MUCI70 and GAUT11 Cause Severe Mucilage Defects 225
To investigate the biological role of MUCI70 in SCE cells we obtained two independent T-226
DNA lines and identified homozygous mutants (Fig 2A Supplemental Table S1) While GATL5 227
was unambiguously shown to be required for mucilage pectin structure (Kong et al 2013) only 228
one of two transcriptional knockdown mutants (gaut11-2 Fig 2A) previously indicated that 229
GAUT11 influences mucilage structure (Caffall et al 2009) Therefore we analyzed two muci70 230
insertional mutants alongside two gaut11 mutants gaut11-3 and gaut11-4 with insertions in 231
GAUT11 exons (Fig 2A) Using reverse transcription quantitative polymerase chain reaction 232
(RT-qPCR) we validated that both MUCI70 and GAUT11 were transcribed in developing 233
Arabidopsis siliques from 3 to 10 days post anthesis (DPA) GAUT11 showed a dramatic 234
increase (around 15-fold) in expression at 7 DPA when pectin synthesis in SCE cells is at its 235
peak (Fig 2B) Based on the results of public microarray datasets (Winter et al 2007 Belmonte 236
et al 2013) both genes were preferentially expressed in the seed coat relative to the embryo 237
(Fig 2C) and had similar transcript levels from the heart stage (~3 DPA) to the mature green 238
stage (~10 DPA) Each insertion in the MUCI70 gene reduced its expression by at least 60 239
(Fig 2D) Although gaut11-3 and gaut11-4 did not significantly alter GAUT11 transcription at 240
either the 5 or 3 end (Fig 2D) these alleles and the previously described gaut11-2 (Caffall et 241
al 2009) are exonic insertions (Fig 2A) that likely disrupt the GAUT11 protein sequence 242
In contrast to wild-type seeds which are surrounded by large mucilage capsules (Fig 3A) 243
two muci70 and two gaut11 homozygous mutants showed severe ruthenium red (RR) staining 244
defects (Fig 3B to 3E) consisting of patchy or completely impaired mucilage release 245
Consequently the muci70-1 muci70-2 and gaut11-3 seeds were surrounded by significantly 246
smaller mucilage capsules (Fig 2E) whose surface area was only 19 to 39 of the wild-type 247
value At least 65 of muci70 and gaut11 seeds floated on water (Fig 2F Fig 3 marked by 248
stars) whereas wild-type seeds did not float (Fig 2F) despite having similar dimensions (Fig 249
2E) Besides GAUT11 three other GAUT genes (GAUT8 GAUT10 GAUT14) were expressed 250
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9
in the developing seed coat (Supplemental Fig S1) The gaut8 mutant was previously found to 251
be lethal but the gaut10-1 and gaut14-1 transcriptional knockout mutants were viable (Caffall et 252
al 2009) and re-examined in this study In contrast to muci70 and gaut11 mutants gaut10-1 253
and gaut14-1 did not disrupt the dimensions of the seeds or the surrounding RR-stained 254
mucilage capsules (Fig 2E Supplemental Fig S2) Therefore only one of the GAUT genes 255
tested was essential on its own for maintaining mucilage architecture consistent with the 256
previous study of the whole GAUT family (Caffall et al 2009) 257
Since both gaut11-3 and gaut11-4 mutants showed similar mucilage staining defects to the 258
previously described gaut11-2 allele (Caffall et al 2009) we primarily used gaut11-3 for further 259
experiments To investigate if MUCI70 and GAUT11 function in the same pathway we crossed 260
the muci70-1 and gaut11-3 single mutants While muci70 and gaut11 single mutants showed 261
smaller RR-stained mucilage capsules than the wild type (Fig 3A to 3E) all muci70 gaut11 262
double mutant seeds failed to release mucilage (Fig 3F) and thus floated on water (Fig 2F) 263
Despite the severe mucilage defects the muci70 gaut11 seeds were only 6 smaller than the 264
wild type (Fig 2E) This suggested that both MUCI70 and GAUT11 might be required for the 265
biosynthesis of pectin in SCE cells which is ultimately released as a hydrophilic capsule from 266
mature seeds 267
268
MUCI70 and GAUT11 Are Important for Pectin Production in SCE Cells 269
To identify the underlying biochemical defects that lead to impaired mucilage release total 270
mucilage was extracted from seeds vigorously mixed using a ball mill (Voiniciuc et al 2015b 271
Voiniciuc and Guumlnl 2016) As previously described this intensive mechanical agitation 272
effectively removes all mucilage polysaccharides resulting in seeds that are no longer stained 273
by RR (Fig 4A) The monosaccharide composition of the total mucilage extracted from hydrated 274
seeds was quantified using high-performance anion-exchange chromatography with pulsed 275
amperometric detection (HPAEC-PAD Supplemental Table S2) Rha and GalA the building 276
blocks of the RG I backbone represent around 90 of total mucilage extracted from wild-type 277
Arabidopsis seeds (Fig 4 Voiniciuc et al 2015c) The muci70-1 and muci70-2 mutations 278
reduced the absolute levels of Rha and GalA in total mucilage extracts by more than 50 279
compared to the wild-type control (Fig 4B) The gaut11-3 single mutant reduced pectin content 280
by around 30 compared to the wild type (Fig 4B) similar to the gaut11-4 allele (Supplemental 281
Table S2) Interestingly the absolute abundance of the minor sugars in the total mucilage 282
extracts increased by more than 40 in the muci70 and gaut11 mutants compared to wild type 283
(Supplemental Table S2) This suggests that both MUCI70 and GAUT11 are particularly 284
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10
important for the production and release of RG I but are not indispensable for the release of the 285
minor mucilage components For comparison a knockout insertion in the MYB5 transcription 286
factor which promotes seed coat differentiation and mucilage production (Li et al 2009 287
Voiniciuc et al 2015c) significantly decreased the content of all sugars found in total mucilage 288
extracts (Supplemental Table S2) Therefore muci70 and gaut11 mutants are deficient in the 289
production and release of pectic polysaccharides In contrast to the gaut11-3 and gaut11-4 290
mutants the gaut10-1 and gaut14-1 knockout mutants identified by genotyping (Supplemental 291
Table S3) reduced Rha and GalA levels by only 8 to 13 (Supplemental Table S2) Consistent 292
with their normal RR staining phenotypes (Supplemental Fig S2) gaut10-1 and gaut14-1 thus 293
had a relatively minor influence on mucilage production Indeed analysis of the muci70-1 294
gaut11-3 double revealed that MUCI70 together with GAUT11 accounted for the biosynthesis 295
and release of 88 of GalA-containing polymers in total seed mucilage extracts (Fig 4B) Two-296
factor ANOVA analysis of the HPAEC-PAD data (Supplemental Table S4) indicated that the 297
muci70-1 and gaut11-3 mutations had purely additive effects on GalA abundance Since the 298
muci70-1 gaut11-3 total mucilage extracts also contained 84 less Rha than the wild type the 299
mutated genes controlled the content of mucilage pectin in a non-redundant manner (Fig 4B) 300
Compared to the single mutants the muci70-1 gaut11-3 double mutant released even more 301
minor sugars in total mucilage extracts (Supplemental Table S2) Since the minor sugars are 302
primarily derived from hemicelluloses (Voiniciuc et al 2015a Voiniciuc et al 2015b) the 303
observed chemotype is consistent with the specific loss of pectin 304
Besides the drastic deficiency of RG I backbone sugars mutations in MUCI70 and GAUT11 305
significantly increased the absolute amounts of Gal Glc and Man in total mucilage extracts (Fig 306
4B Supplemental Table S2) but had distinct effects on the content of Ara and Xyl Based on 307
ANOVA the muci70-1 and gaut11-3 mutations had purely additive effects on the content of Gal 308
while the increases in Glc and Man content were higher than expected (Supplemental Table 309
S4) The two muci70 alleles significantly decreased Ara content (26 to 32) relative to the wild 310
type and gaut11 mutants ANOVA confirmed that only MUCI70 influenced the presence of Ara 311
(Supplemental Table S4) Surprisingly muci70 and gaut11 single mutants had polarizing effects 312
on Xyl content Relative to wild type muci70 single mutants increased Xyl abundance by 73 to 313
87 while the gaut11 single mutants and the muci70-1 gaut11-3 double mutant decreased Xyl 314
content by 43 to 47 (Supplemental Table S2) 315
To further investigate the structure of pectin and other polysaccharides glycosyl linkage 316
analysis was performed on total mucilage extracts (Table I) Relative to the wild-type control the 317
total mucilage extracts of both the muci70-1 and gaut11-3 mutants contained significant 318
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11
reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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12
sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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13
expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
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693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
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stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
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24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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- Parsed Citations
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- Figure 1
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6
tissues and that the relevant GTs are not part of classified CAZy families (Harholt et al 2010) 150
These obstacles were surpassed by the identification of novel GT-like plant proteins through 151
Golgi proteomic studies (Nikolovski et al 2012 Nikolovski et al 2014) and the establishment 152
of Arabidopsis seed mucilage as a model for dissecting pectin synthesis (Haughn and Western 153
2012) Within a narrow developmental window Arabidopsis seed coat epidermal (SCE) cells 154
produce copious amounts of RG I along with minor amounts of cellulose hemicellulose 155
arabinogalactans and HG (Voiniciuc et al 2015c) Since at least 90 of the mucilage 156
extracted from Arabidopsis seeds consists of Rha and GalA units derived from pectin the SCE 157
cells can be exploited to identify pectin-related GTs In addition structural changes in seed 158
mucilage polysaccharides can be conveniently monitored in situ with a variety of imaging 159
techniques and specific probes (Voiniciuc et al 2018) 160
Despite the great potential of this model system only two GTs have been implicated so 161
far in the synthesis of the pectin domains in mucilage A screen of 26 gaut mutant lines for 162
altered staining of seed mucilage found only one mutant (gaut11-2) that showed smaller 163
mucilage capsules and reduced uronic acid content compared to the wild type (Caffall et al 164
2009) Although the results indicated that GAUT11 might affect HG biosynthesis in SCE cells 165
the gaut11-2 phenotype was not supported by an independent knockdown gaut11-1 allele 166
(Caffall et al 2009) GATL5 another protein from the GT8 family is the only other pectin-167
related GT that has been implicated in mucilage biosynthesis A knockout T-DNA insertion in 168
GATL5 increased the molecular weight of mucilage polysaccharides without dramatically 169
altering the glycosidic linkage composition or the content of pectin epitopes bound by antibodies 170
(Kong et al 2013) Since GATL5 was proposed to simply regulate the final size of pectin 171
polymers in mucilage additional players must be required for the elongation of RG I in 172
Arabidopsis SCE cells 173
Recently a co-expression and sequence-based MUCILAGE-RELATED (MUCI) reverse 174
genetic screen identified three GTs required for the synthesis of two distinct hemicellulosic 175
polymers (xylan and galactoglucomannan) in Arabidopsis SCE cells (Voiniciuc et al 2015b 176
Voiniciuc et al 2015a) Using an extension of this strategy we now report that the biosynthesis 177
of pectin requires MUCI70 a putative GT from an unclassified CAZy family that was not known 178
to affect cell wall structure Through a detailed biochemical and histological characterization of 179
muci70 mutants and two novel gaut11 alleles we show that these two genes are required for 180
the production of two distinct RG I domains essential for seed mucilage architecture Finally the 181
analysis of a muci70 gaut11 double mutant and the demonstration that GAUT11 is an HG α-182
GalA transferase confirms that MUCI70 and GAUT11 are indispensable for the production of 183
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7
two RG I domains that represent the bulk of seed mucilage and shape the surface morphology 184
of seeds 185
186
RESULTS 187
MUCI70 is a Novel Pectin-Related GT Localized in the Golgi apparatus 188
To identify novel players involved in pectin production we systematically profiled the expression 189
of all 1128 Arabidopsis thaliana members of the CAZy database (Lombard et al 2014) in the 190
seed coat using ATH1 microarray data in Genevestigator (Hruz et al 2008) This strategy 191
revealed more than 50 CAZy genes that are transcribed in the seed coat when mucilage is 192
produced The majority of these genes were not identified in the initial MUCI screen (Voiniciuc 193
et al 2015b) because they are not significantly co-expressed with known mucilage genes in 194
GeneCAT (Mutwil et al 2008) GeneMANIA (Warde-Farley et al 2010) and ATTED-II 195
(Obayashi et al 2014) Amongst this collection of genes were MUCI64IRX14 (Voiniciuc et al 196
2015a) and four members of the GAUT family (Supplemental Fig S1) including GAUT11 and 197
GATL5 Interestingly we also found one gene encoding a putative GT (At1g28240) which we 198
named MUCI70 as a promising candidate for pectin production in the Arabidopsis seed coat 199
MUCI70 represents the founding member of a GT family whose roles in cell wall biology 200
remain unclear (Fig 1A) The MUCI70 protein contains a single transmembrane domain 201
(AREMEMNON Consensus TM α-helix prediction AramTmConl Schwacke et al 2003) near its 202
N-terminus and a DUF616 (PF04765) conserved domain of unknown function (Fig 1B) 203
Phylogenetic analysis of DUF616 proteins organized MUCI70 and its six Arabidopsis paralogs 204
into four clades (Fig 1A) Each of these groups contains at least one ortholog in both 205
Physcomitrella patens and Selaginella moellendorffii members of two early diverging lineages 206
of land plants (Fig 1A) In contrast TURGOR REGULATION DEFECT 1 (TOD1 AT5G46220) 207
the only other Arabidopsis protein containing a DUF616 motif did not cluster with any of these 208
clades (Fig 1A) and appeared to be functionally distinct Indeed TOD1 was demonstrated to 209
have alkaline ceramidase activity in vitro (Chen et al 2015) rather than a CAZy-related 210
function At4g38500 a close paralog of MUCI70 (Fig 1A) was previously identified in a Golgi 211
proteomics study and showed little similarity in primary sequence and predicted 3D structure to 212
the GT8 family in Arabidopsis (Nikolovski et al 2012) Based on tight co-expression with GAUT 213
genes At4g38500 was hypothesized to be involved in pectin biosynthesis (Voxeur et al 2012) 214
MUCI70 and GAUT11 a gene that was implicated in mucilage HG biosynthesis (Caffall et 215
al 2009) showed similar transcriptional profiles in developing seeds (Supplemental Fig S1 216
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8
Belmonte et al 2013) and encode proteins with similar topologies (Fig 1B) GAUT11 was 217
previously found in the Golgi proteome (Parsons et al 2012) but the subcellular localization of 218
MUCI70 remained unknown To address this MUCI70 tagged with super yellow fluorescent 219
protein (sYFP) was stably expressed in Arabidopsis using the constitutive 35S promoter 220
MUCI70-sYFP was observed in intracellular punctae (Fig 1C) that co-localized with the Golgi 221
marker sialyltransferase (ST) tagged with red fluorescent protein (ST-RFP Figs 1D and 1E) 222
which marks the site of pectin production in plants 223
224
Mutations in MUCI70 and GAUT11 Cause Severe Mucilage Defects 225
To investigate the biological role of MUCI70 in SCE cells we obtained two independent T-226
DNA lines and identified homozygous mutants (Fig 2A Supplemental Table S1) While GATL5 227
was unambiguously shown to be required for mucilage pectin structure (Kong et al 2013) only 228
one of two transcriptional knockdown mutants (gaut11-2 Fig 2A) previously indicated that 229
GAUT11 influences mucilage structure (Caffall et al 2009) Therefore we analyzed two muci70 230
insertional mutants alongside two gaut11 mutants gaut11-3 and gaut11-4 with insertions in 231
GAUT11 exons (Fig 2A) Using reverse transcription quantitative polymerase chain reaction 232
(RT-qPCR) we validated that both MUCI70 and GAUT11 were transcribed in developing 233
Arabidopsis siliques from 3 to 10 days post anthesis (DPA) GAUT11 showed a dramatic 234
increase (around 15-fold) in expression at 7 DPA when pectin synthesis in SCE cells is at its 235
peak (Fig 2B) Based on the results of public microarray datasets (Winter et al 2007 Belmonte 236
et al 2013) both genes were preferentially expressed in the seed coat relative to the embryo 237
(Fig 2C) and had similar transcript levels from the heart stage (~3 DPA) to the mature green 238
stage (~10 DPA) Each insertion in the MUCI70 gene reduced its expression by at least 60 239
(Fig 2D) Although gaut11-3 and gaut11-4 did not significantly alter GAUT11 transcription at 240
either the 5 or 3 end (Fig 2D) these alleles and the previously described gaut11-2 (Caffall et 241
al 2009) are exonic insertions (Fig 2A) that likely disrupt the GAUT11 protein sequence 242
In contrast to wild-type seeds which are surrounded by large mucilage capsules (Fig 3A) 243
two muci70 and two gaut11 homozygous mutants showed severe ruthenium red (RR) staining 244
defects (Fig 3B to 3E) consisting of patchy or completely impaired mucilage release 245
Consequently the muci70-1 muci70-2 and gaut11-3 seeds were surrounded by significantly 246
smaller mucilage capsules (Fig 2E) whose surface area was only 19 to 39 of the wild-type 247
value At least 65 of muci70 and gaut11 seeds floated on water (Fig 2F Fig 3 marked by 248
stars) whereas wild-type seeds did not float (Fig 2F) despite having similar dimensions (Fig 249
2E) Besides GAUT11 three other GAUT genes (GAUT8 GAUT10 GAUT14) were expressed 250
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9
in the developing seed coat (Supplemental Fig S1) The gaut8 mutant was previously found to 251
be lethal but the gaut10-1 and gaut14-1 transcriptional knockout mutants were viable (Caffall et 252
al 2009) and re-examined in this study In contrast to muci70 and gaut11 mutants gaut10-1 253
and gaut14-1 did not disrupt the dimensions of the seeds or the surrounding RR-stained 254
mucilage capsules (Fig 2E Supplemental Fig S2) Therefore only one of the GAUT genes 255
tested was essential on its own for maintaining mucilage architecture consistent with the 256
previous study of the whole GAUT family (Caffall et al 2009) 257
Since both gaut11-3 and gaut11-4 mutants showed similar mucilage staining defects to the 258
previously described gaut11-2 allele (Caffall et al 2009) we primarily used gaut11-3 for further 259
experiments To investigate if MUCI70 and GAUT11 function in the same pathway we crossed 260
the muci70-1 and gaut11-3 single mutants While muci70 and gaut11 single mutants showed 261
smaller RR-stained mucilage capsules than the wild type (Fig 3A to 3E) all muci70 gaut11 262
double mutant seeds failed to release mucilage (Fig 3F) and thus floated on water (Fig 2F) 263
Despite the severe mucilage defects the muci70 gaut11 seeds were only 6 smaller than the 264
wild type (Fig 2E) This suggested that both MUCI70 and GAUT11 might be required for the 265
biosynthesis of pectin in SCE cells which is ultimately released as a hydrophilic capsule from 266
mature seeds 267
268
MUCI70 and GAUT11 Are Important for Pectin Production in SCE Cells 269
To identify the underlying biochemical defects that lead to impaired mucilage release total 270
mucilage was extracted from seeds vigorously mixed using a ball mill (Voiniciuc et al 2015b 271
Voiniciuc and Guumlnl 2016) As previously described this intensive mechanical agitation 272
effectively removes all mucilage polysaccharides resulting in seeds that are no longer stained 273
by RR (Fig 4A) The monosaccharide composition of the total mucilage extracted from hydrated 274
seeds was quantified using high-performance anion-exchange chromatography with pulsed 275
amperometric detection (HPAEC-PAD Supplemental Table S2) Rha and GalA the building 276
blocks of the RG I backbone represent around 90 of total mucilage extracted from wild-type 277
Arabidopsis seeds (Fig 4 Voiniciuc et al 2015c) The muci70-1 and muci70-2 mutations 278
reduced the absolute levels of Rha and GalA in total mucilage extracts by more than 50 279
compared to the wild-type control (Fig 4B) The gaut11-3 single mutant reduced pectin content 280
by around 30 compared to the wild type (Fig 4B) similar to the gaut11-4 allele (Supplemental 281
Table S2) Interestingly the absolute abundance of the minor sugars in the total mucilage 282
extracts increased by more than 40 in the muci70 and gaut11 mutants compared to wild type 283
(Supplemental Table S2) This suggests that both MUCI70 and GAUT11 are particularly 284
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10
important for the production and release of RG I but are not indispensable for the release of the 285
minor mucilage components For comparison a knockout insertion in the MYB5 transcription 286
factor which promotes seed coat differentiation and mucilage production (Li et al 2009 287
Voiniciuc et al 2015c) significantly decreased the content of all sugars found in total mucilage 288
extracts (Supplemental Table S2) Therefore muci70 and gaut11 mutants are deficient in the 289
production and release of pectic polysaccharides In contrast to the gaut11-3 and gaut11-4 290
mutants the gaut10-1 and gaut14-1 knockout mutants identified by genotyping (Supplemental 291
Table S3) reduced Rha and GalA levels by only 8 to 13 (Supplemental Table S2) Consistent 292
with their normal RR staining phenotypes (Supplemental Fig S2) gaut10-1 and gaut14-1 thus 293
had a relatively minor influence on mucilage production Indeed analysis of the muci70-1 294
gaut11-3 double revealed that MUCI70 together with GAUT11 accounted for the biosynthesis 295
and release of 88 of GalA-containing polymers in total seed mucilage extracts (Fig 4B) Two-296
factor ANOVA analysis of the HPAEC-PAD data (Supplemental Table S4) indicated that the 297
muci70-1 and gaut11-3 mutations had purely additive effects on GalA abundance Since the 298
muci70-1 gaut11-3 total mucilage extracts also contained 84 less Rha than the wild type the 299
mutated genes controlled the content of mucilage pectin in a non-redundant manner (Fig 4B) 300
Compared to the single mutants the muci70-1 gaut11-3 double mutant released even more 301
minor sugars in total mucilage extracts (Supplemental Table S2) Since the minor sugars are 302
primarily derived from hemicelluloses (Voiniciuc et al 2015a Voiniciuc et al 2015b) the 303
observed chemotype is consistent with the specific loss of pectin 304
Besides the drastic deficiency of RG I backbone sugars mutations in MUCI70 and GAUT11 305
significantly increased the absolute amounts of Gal Glc and Man in total mucilage extracts (Fig 306
4B Supplemental Table S2) but had distinct effects on the content of Ara and Xyl Based on 307
ANOVA the muci70-1 and gaut11-3 mutations had purely additive effects on the content of Gal 308
while the increases in Glc and Man content were higher than expected (Supplemental Table 309
S4) The two muci70 alleles significantly decreased Ara content (26 to 32) relative to the wild 310
type and gaut11 mutants ANOVA confirmed that only MUCI70 influenced the presence of Ara 311
(Supplemental Table S4) Surprisingly muci70 and gaut11 single mutants had polarizing effects 312
on Xyl content Relative to wild type muci70 single mutants increased Xyl abundance by 73 to 313
87 while the gaut11 single mutants and the muci70-1 gaut11-3 double mutant decreased Xyl 314
content by 43 to 47 (Supplemental Table S2) 315
To further investigate the structure of pectin and other polysaccharides glycosyl linkage 316
analysis was performed on total mucilage extracts (Table I) Relative to the wild-type control the 317
total mucilage extracts of both the muci70-1 and gaut11-3 mutants contained significant 318
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11
reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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7
two RG I domains that represent the bulk of seed mucilage and shape the surface morphology 184
of seeds 185
186
RESULTS 187
MUCI70 is a Novel Pectin-Related GT Localized in the Golgi apparatus 188
To identify novel players involved in pectin production we systematically profiled the expression 189
of all 1128 Arabidopsis thaliana members of the CAZy database (Lombard et al 2014) in the 190
seed coat using ATH1 microarray data in Genevestigator (Hruz et al 2008) This strategy 191
revealed more than 50 CAZy genes that are transcribed in the seed coat when mucilage is 192
produced The majority of these genes were not identified in the initial MUCI screen (Voiniciuc 193
et al 2015b) because they are not significantly co-expressed with known mucilage genes in 194
GeneCAT (Mutwil et al 2008) GeneMANIA (Warde-Farley et al 2010) and ATTED-II 195
(Obayashi et al 2014) Amongst this collection of genes were MUCI64IRX14 (Voiniciuc et al 196
2015a) and four members of the GAUT family (Supplemental Fig S1) including GAUT11 and 197
GATL5 Interestingly we also found one gene encoding a putative GT (At1g28240) which we 198
named MUCI70 as a promising candidate for pectin production in the Arabidopsis seed coat 199
MUCI70 represents the founding member of a GT family whose roles in cell wall biology 200
remain unclear (Fig 1A) The MUCI70 protein contains a single transmembrane domain 201
(AREMEMNON Consensus TM α-helix prediction AramTmConl Schwacke et al 2003) near its 202
N-terminus and a DUF616 (PF04765) conserved domain of unknown function (Fig 1B) 203
Phylogenetic analysis of DUF616 proteins organized MUCI70 and its six Arabidopsis paralogs 204
into four clades (Fig 1A) Each of these groups contains at least one ortholog in both 205
Physcomitrella patens and Selaginella moellendorffii members of two early diverging lineages 206
of land plants (Fig 1A) In contrast TURGOR REGULATION DEFECT 1 (TOD1 AT5G46220) 207
the only other Arabidopsis protein containing a DUF616 motif did not cluster with any of these 208
clades (Fig 1A) and appeared to be functionally distinct Indeed TOD1 was demonstrated to 209
have alkaline ceramidase activity in vitro (Chen et al 2015) rather than a CAZy-related 210
function At4g38500 a close paralog of MUCI70 (Fig 1A) was previously identified in a Golgi 211
proteomics study and showed little similarity in primary sequence and predicted 3D structure to 212
the GT8 family in Arabidopsis (Nikolovski et al 2012) Based on tight co-expression with GAUT 213
genes At4g38500 was hypothesized to be involved in pectin biosynthesis (Voxeur et al 2012) 214
MUCI70 and GAUT11 a gene that was implicated in mucilage HG biosynthesis (Caffall et 215
al 2009) showed similar transcriptional profiles in developing seeds (Supplemental Fig S1 216
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
Belmonte et al 2013) and encode proteins with similar topologies (Fig 1B) GAUT11 was 217
previously found in the Golgi proteome (Parsons et al 2012) but the subcellular localization of 218
MUCI70 remained unknown To address this MUCI70 tagged with super yellow fluorescent 219
protein (sYFP) was stably expressed in Arabidopsis using the constitutive 35S promoter 220
MUCI70-sYFP was observed in intracellular punctae (Fig 1C) that co-localized with the Golgi 221
marker sialyltransferase (ST) tagged with red fluorescent protein (ST-RFP Figs 1D and 1E) 222
which marks the site of pectin production in plants 223
224
Mutations in MUCI70 and GAUT11 Cause Severe Mucilage Defects 225
To investigate the biological role of MUCI70 in SCE cells we obtained two independent T-226
DNA lines and identified homozygous mutants (Fig 2A Supplemental Table S1) While GATL5 227
was unambiguously shown to be required for mucilage pectin structure (Kong et al 2013) only 228
one of two transcriptional knockdown mutants (gaut11-2 Fig 2A) previously indicated that 229
GAUT11 influences mucilage structure (Caffall et al 2009) Therefore we analyzed two muci70 230
insertional mutants alongside two gaut11 mutants gaut11-3 and gaut11-4 with insertions in 231
GAUT11 exons (Fig 2A) Using reverse transcription quantitative polymerase chain reaction 232
(RT-qPCR) we validated that both MUCI70 and GAUT11 were transcribed in developing 233
Arabidopsis siliques from 3 to 10 days post anthesis (DPA) GAUT11 showed a dramatic 234
increase (around 15-fold) in expression at 7 DPA when pectin synthesis in SCE cells is at its 235
peak (Fig 2B) Based on the results of public microarray datasets (Winter et al 2007 Belmonte 236
et al 2013) both genes were preferentially expressed in the seed coat relative to the embryo 237
(Fig 2C) and had similar transcript levels from the heart stage (~3 DPA) to the mature green 238
stage (~10 DPA) Each insertion in the MUCI70 gene reduced its expression by at least 60 239
(Fig 2D) Although gaut11-3 and gaut11-4 did not significantly alter GAUT11 transcription at 240
either the 5 or 3 end (Fig 2D) these alleles and the previously described gaut11-2 (Caffall et 241
al 2009) are exonic insertions (Fig 2A) that likely disrupt the GAUT11 protein sequence 242
In contrast to wild-type seeds which are surrounded by large mucilage capsules (Fig 3A) 243
two muci70 and two gaut11 homozygous mutants showed severe ruthenium red (RR) staining 244
defects (Fig 3B to 3E) consisting of patchy or completely impaired mucilage release 245
Consequently the muci70-1 muci70-2 and gaut11-3 seeds were surrounded by significantly 246
smaller mucilage capsules (Fig 2E) whose surface area was only 19 to 39 of the wild-type 247
value At least 65 of muci70 and gaut11 seeds floated on water (Fig 2F Fig 3 marked by 248
stars) whereas wild-type seeds did not float (Fig 2F) despite having similar dimensions (Fig 249
2E) Besides GAUT11 three other GAUT genes (GAUT8 GAUT10 GAUT14) were expressed 250
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
in the developing seed coat (Supplemental Fig S1) The gaut8 mutant was previously found to 251
be lethal but the gaut10-1 and gaut14-1 transcriptional knockout mutants were viable (Caffall et 252
al 2009) and re-examined in this study In contrast to muci70 and gaut11 mutants gaut10-1 253
and gaut14-1 did not disrupt the dimensions of the seeds or the surrounding RR-stained 254
mucilage capsules (Fig 2E Supplemental Fig S2) Therefore only one of the GAUT genes 255
tested was essential on its own for maintaining mucilage architecture consistent with the 256
previous study of the whole GAUT family (Caffall et al 2009) 257
Since both gaut11-3 and gaut11-4 mutants showed similar mucilage staining defects to the 258
previously described gaut11-2 allele (Caffall et al 2009) we primarily used gaut11-3 for further 259
experiments To investigate if MUCI70 and GAUT11 function in the same pathway we crossed 260
the muci70-1 and gaut11-3 single mutants While muci70 and gaut11 single mutants showed 261
smaller RR-stained mucilage capsules than the wild type (Fig 3A to 3E) all muci70 gaut11 262
double mutant seeds failed to release mucilage (Fig 3F) and thus floated on water (Fig 2F) 263
Despite the severe mucilage defects the muci70 gaut11 seeds were only 6 smaller than the 264
wild type (Fig 2E) This suggested that both MUCI70 and GAUT11 might be required for the 265
biosynthesis of pectin in SCE cells which is ultimately released as a hydrophilic capsule from 266
mature seeds 267
268
MUCI70 and GAUT11 Are Important for Pectin Production in SCE Cells 269
To identify the underlying biochemical defects that lead to impaired mucilage release total 270
mucilage was extracted from seeds vigorously mixed using a ball mill (Voiniciuc et al 2015b 271
Voiniciuc and Guumlnl 2016) As previously described this intensive mechanical agitation 272
effectively removes all mucilage polysaccharides resulting in seeds that are no longer stained 273
by RR (Fig 4A) The monosaccharide composition of the total mucilage extracted from hydrated 274
seeds was quantified using high-performance anion-exchange chromatography with pulsed 275
amperometric detection (HPAEC-PAD Supplemental Table S2) Rha and GalA the building 276
blocks of the RG I backbone represent around 90 of total mucilage extracted from wild-type 277
Arabidopsis seeds (Fig 4 Voiniciuc et al 2015c) The muci70-1 and muci70-2 mutations 278
reduced the absolute levels of Rha and GalA in total mucilage extracts by more than 50 279
compared to the wild-type control (Fig 4B) The gaut11-3 single mutant reduced pectin content 280
by around 30 compared to the wild type (Fig 4B) similar to the gaut11-4 allele (Supplemental 281
Table S2) Interestingly the absolute abundance of the minor sugars in the total mucilage 282
extracts increased by more than 40 in the muci70 and gaut11 mutants compared to wild type 283
(Supplemental Table S2) This suggests that both MUCI70 and GAUT11 are particularly 284
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10
important for the production and release of RG I but are not indispensable for the release of the 285
minor mucilage components For comparison a knockout insertion in the MYB5 transcription 286
factor which promotes seed coat differentiation and mucilage production (Li et al 2009 287
Voiniciuc et al 2015c) significantly decreased the content of all sugars found in total mucilage 288
extracts (Supplemental Table S2) Therefore muci70 and gaut11 mutants are deficient in the 289
production and release of pectic polysaccharides In contrast to the gaut11-3 and gaut11-4 290
mutants the gaut10-1 and gaut14-1 knockout mutants identified by genotyping (Supplemental 291
Table S3) reduced Rha and GalA levels by only 8 to 13 (Supplemental Table S2) Consistent 292
with their normal RR staining phenotypes (Supplemental Fig S2) gaut10-1 and gaut14-1 thus 293
had a relatively minor influence on mucilage production Indeed analysis of the muci70-1 294
gaut11-3 double revealed that MUCI70 together with GAUT11 accounted for the biosynthesis 295
and release of 88 of GalA-containing polymers in total seed mucilage extracts (Fig 4B) Two-296
factor ANOVA analysis of the HPAEC-PAD data (Supplemental Table S4) indicated that the 297
muci70-1 and gaut11-3 mutations had purely additive effects on GalA abundance Since the 298
muci70-1 gaut11-3 total mucilage extracts also contained 84 less Rha than the wild type the 299
mutated genes controlled the content of mucilage pectin in a non-redundant manner (Fig 4B) 300
Compared to the single mutants the muci70-1 gaut11-3 double mutant released even more 301
minor sugars in total mucilage extracts (Supplemental Table S2) Since the minor sugars are 302
primarily derived from hemicelluloses (Voiniciuc et al 2015a Voiniciuc et al 2015b) the 303
observed chemotype is consistent with the specific loss of pectin 304
Besides the drastic deficiency of RG I backbone sugars mutations in MUCI70 and GAUT11 305
significantly increased the absolute amounts of Gal Glc and Man in total mucilage extracts (Fig 306
4B Supplemental Table S2) but had distinct effects on the content of Ara and Xyl Based on 307
ANOVA the muci70-1 and gaut11-3 mutations had purely additive effects on the content of Gal 308
while the increases in Glc and Man content were higher than expected (Supplemental Table 309
S4) The two muci70 alleles significantly decreased Ara content (26 to 32) relative to the wild 310
type and gaut11 mutants ANOVA confirmed that only MUCI70 influenced the presence of Ara 311
(Supplemental Table S4) Surprisingly muci70 and gaut11 single mutants had polarizing effects 312
on Xyl content Relative to wild type muci70 single mutants increased Xyl abundance by 73 to 313
87 while the gaut11 single mutants and the muci70-1 gaut11-3 double mutant decreased Xyl 314
content by 43 to 47 (Supplemental Table S2) 315
To further investigate the structure of pectin and other polysaccharides glycosyl linkage 316
analysis was performed on total mucilage extracts (Table I) Relative to the wild-type control the 317
total mucilage extracts of both the muci70-1 and gaut11-3 mutants contained significant 318
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reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
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27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
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28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
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29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
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30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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8
Belmonte et al 2013) and encode proteins with similar topologies (Fig 1B) GAUT11 was 217
previously found in the Golgi proteome (Parsons et al 2012) but the subcellular localization of 218
MUCI70 remained unknown To address this MUCI70 tagged with super yellow fluorescent 219
protein (sYFP) was stably expressed in Arabidopsis using the constitutive 35S promoter 220
MUCI70-sYFP was observed in intracellular punctae (Fig 1C) that co-localized with the Golgi 221
marker sialyltransferase (ST) tagged with red fluorescent protein (ST-RFP Figs 1D and 1E) 222
which marks the site of pectin production in plants 223
224
Mutations in MUCI70 and GAUT11 Cause Severe Mucilage Defects 225
To investigate the biological role of MUCI70 in SCE cells we obtained two independent T-226
DNA lines and identified homozygous mutants (Fig 2A Supplemental Table S1) While GATL5 227
was unambiguously shown to be required for mucilage pectin structure (Kong et al 2013) only 228
one of two transcriptional knockdown mutants (gaut11-2 Fig 2A) previously indicated that 229
GAUT11 influences mucilage structure (Caffall et al 2009) Therefore we analyzed two muci70 230
insertional mutants alongside two gaut11 mutants gaut11-3 and gaut11-4 with insertions in 231
GAUT11 exons (Fig 2A) Using reverse transcription quantitative polymerase chain reaction 232
(RT-qPCR) we validated that both MUCI70 and GAUT11 were transcribed in developing 233
Arabidopsis siliques from 3 to 10 days post anthesis (DPA) GAUT11 showed a dramatic 234
increase (around 15-fold) in expression at 7 DPA when pectin synthesis in SCE cells is at its 235
peak (Fig 2B) Based on the results of public microarray datasets (Winter et al 2007 Belmonte 236
et al 2013) both genes were preferentially expressed in the seed coat relative to the embryo 237
(Fig 2C) and had similar transcript levels from the heart stage (~3 DPA) to the mature green 238
stage (~10 DPA) Each insertion in the MUCI70 gene reduced its expression by at least 60 239
(Fig 2D) Although gaut11-3 and gaut11-4 did not significantly alter GAUT11 transcription at 240
either the 5 or 3 end (Fig 2D) these alleles and the previously described gaut11-2 (Caffall et 241
al 2009) are exonic insertions (Fig 2A) that likely disrupt the GAUT11 protein sequence 242
In contrast to wild-type seeds which are surrounded by large mucilage capsules (Fig 3A) 243
two muci70 and two gaut11 homozygous mutants showed severe ruthenium red (RR) staining 244
defects (Fig 3B to 3E) consisting of patchy or completely impaired mucilage release 245
Consequently the muci70-1 muci70-2 and gaut11-3 seeds were surrounded by significantly 246
smaller mucilage capsules (Fig 2E) whose surface area was only 19 to 39 of the wild-type 247
value At least 65 of muci70 and gaut11 seeds floated on water (Fig 2F Fig 3 marked by 248
stars) whereas wild-type seeds did not float (Fig 2F) despite having similar dimensions (Fig 249
2E) Besides GAUT11 three other GAUT genes (GAUT8 GAUT10 GAUT14) were expressed 250
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
in the developing seed coat (Supplemental Fig S1) The gaut8 mutant was previously found to 251
be lethal but the gaut10-1 and gaut14-1 transcriptional knockout mutants were viable (Caffall et 252
al 2009) and re-examined in this study In contrast to muci70 and gaut11 mutants gaut10-1 253
and gaut14-1 did not disrupt the dimensions of the seeds or the surrounding RR-stained 254
mucilage capsules (Fig 2E Supplemental Fig S2) Therefore only one of the GAUT genes 255
tested was essential on its own for maintaining mucilage architecture consistent with the 256
previous study of the whole GAUT family (Caffall et al 2009) 257
Since both gaut11-3 and gaut11-4 mutants showed similar mucilage staining defects to the 258
previously described gaut11-2 allele (Caffall et al 2009) we primarily used gaut11-3 for further 259
experiments To investigate if MUCI70 and GAUT11 function in the same pathway we crossed 260
the muci70-1 and gaut11-3 single mutants While muci70 and gaut11 single mutants showed 261
smaller RR-stained mucilage capsules than the wild type (Fig 3A to 3E) all muci70 gaut11 262
double mutant seeds failed to release mucilage (Fig 3F) and thus floated on water (Fig 2F) 263
Despite the severe mucilage defects the muci70 gaut11 seeds were only 6 smaller than the 264
wild type (Fig 2E) This suggested that both MUCI70 and GAUT11 might be required for the 265
biosynthesis of pectin in SCE cells which is ultimately released as a hydrophilic capsule from 266
mature seeds 267
268
MUCI70 and GAUT11 Are Important for Pectin Production in SCE Cells 269
To identify the underlying biochemical defects that lead to impaired mucilage release total 270
mucilage was extracted from seeds vigorously mixed using a ball mill (Voiniciuc et al 2015b 271
Voiniciuc and Guumlnl 2016) As previously described this intensive mechanical agitation 272
effectively removes all mucilage polysaccharides resulting in seeds that are no longer stained 273
by RR (Fig 4A) The monosaccharide composition of the total mucilage extracted from hydrated 274
seeds was quantified using high-performance anion-exchange chromatography with pulsed 275
amperometric detection (HPAEC-PAD Supplemental Table S2) Rha and GalA the building 276
blocks of the RG I backbone represent around 90 of total mucilage extracted from wild-type 277
Arabidopsis seeds (Fig 4 Voiniciuc et al 2015c) The muci70-1 and muci70-2 mutations 278
reduced the absolute levels of Rha and GalA in total mucilage extracts by more than 50 279
compared to the wild-type control (Fig 4B) The gaut11-3 single mutant reduced pectin content 280
by around 30 compared to the wild type (Fig 4B) similar to the gaut11-4 allele (Supplemental 281
Table S2) Interestingly the absolute abundance of the minor sugars in the total mucilage 282
extracts increased by more than 40 in the muci70 and gaut11 mutants compared to wild type 283
(Supplemental Table S2) This suggests that both MUCI70 and GAUT11 are particularly 284
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important for the production and release of RG I but are not indispensable for the release of the 285
minor mucilage components For comparison a knockout insertion in the MYB5 transcription 286
factor which promotes seed coat differentiation and mucilage production (Li et al 2009 287
Voiniciuc et al 2015c) significantly decreased the content of all sugars found in total mucilage 288
extracts (Supplemental Table S2) Therefore muci70 and gaut11 mutants are deficient in the 289
production and release of pectic polysaccharides In contrast to the gaut11-3 and gaut11-4 290
mutants the gaut10-1 and gaut14-1 knockout mutants identified by genotyping (Supplemental 291
Table S3) reduced Rha and GalA levels by only 8 to 13 (Supplemental Table S2) Consistent 292
with their normal RR staining phenotypes (Supplemental Fig S2) gaut10-1 and gaut14-1 thus 293
had a relatively minor influence on mucilage production Indeed analysis of the muci70-1 294
gaut11-3 double revealed that MUCI70 together with GAUT11 accounted for the biosynthesis 295
and release of 88 of GalA-containing polymers in total seed mucilage extracts (Fig 4B) Two-296
factor ANOVA analysis of the HPAEC-PAD data (Supplemental Table S4) indicated that the 297
muci70-1 and gaut11-3 mutations had purely additive effects on GalA abundance Since the 298
muci70-1 gaut11-3 total mucilage extracts also contained 84 less Rha than the wild type the 299
mutated genes controlled the content of mucilage pectin in a non-redundant manner (Fig 4B) 300
Compared to the single mutants the muci70-1 gaut11-3 double mutant released even more 301
minor sugars in total mucilage extracts (Supplemental Table S2) Since the minor sugars are 302
primarily derived from hemicelluloses (Voiniciuc et al 2015a Voiniciuc et al 2015b) the 303
observed chemotype is consistent with the specific loss of pectin 304
Besides the drastic deficiency of RG I backbone sugars mutations in MUCI70 and GAUT11 305
significantly increased the absolute amounts of Gal Glc and Man in total mucilage extracts (Fig 306
4B Supplemental Table S2) but had distinct effects on the content of Ara and Xyl Based on 307
ANOVA the muci70-1 and gaut11-3 mutations had purely additive effects on the content of Gal 308
while the increases in Glc and Man content were higher than expected (Supplemental Table 309
S4) The two muci70 alleles significantly decreased Ara content (26 to 32) relative to the wild 310
type and gaut11 mutants ANOVA confirmed that only MUCI70 influenced the presence of Ara 311
(Supplemental Table S4) Surprisingly muci70 and gaut11 single mutants had polarizing effects 312
on Xyl content Relative to wild type muci70 single mutants increased Xyl abundance by 73 to 313
87 while the gaut11 single mutants and the muci70-1 gaut11-3 double mutant decreased Xyl 314
content by 43 to 47 (Supplemental Table S2) 315
To further investigate the structure of pectin and other polysaccharides glycosyl linkage 316
analysis was performed on total mucilage extracts (Table I) Relative to the wild-type control the 317
total mucilage extracts of both the muci70-1 and gaut11-3 mutants contained significant 318
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reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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20
providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
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25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
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27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
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28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
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29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
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30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
9
in the developing seed coat (Supplemental Fig S1) The gaut8 mutant was previously found to 251
be lethal but the gaut10-1 and gaut14-1 transcriptional knockout mutants were viable (Caffall et 252
al 2009) and re-examined in this study In contrast to muci70 and gaut11 mutants gaut10-1 253
and gaut14-1 did not disrupt the dimensions of the seeds or the surrounding RR-stained 254
mucilage capsules (Fig 2E Supplemental Fig S2) Therefore only one of the GAUT genes 255
tested was essential on its own for maintaining mucilage architecture consistent with the 256
previous study of the whole GAUT family (Caffall et al 2009) 257
Since both gaut11-3 and gaut11-4 mutants showed similar mucilage staining defects to the 258
previously described gaut11-2 allele (Caffall et al 2009) we primarily used gaut11-3 for further 259
experiments To investigate if MUCI70 and GAUT11 function in the same pathway we crossed 260
the muci70-1 and gaut11-3 single mutants While muci70 and gaut11 single mutants showed 261
smaller RR-stained mucilage capsules than the wild type (Fig 3A to 3E) all muci70 gaut11 262
double mutant seeds failed to release mucilage (Fig 3F) and thus floated on water (Fig 2F) 263
Despite the severe mucilage defects the muci70 gaut11 seeds were only 6 smaller than the 264
wild type (Fig 2E) This suggested that both MUCI70 and GAUT11 might be required for the 265
biosynthesis of pectin in SCE cells which is ultimately released as a hydrophilic capsule from 266
mature seeds 267
268
MUCI70 and GAUT11 Are Important for Pectin Production in SCE Cells 269
To identify the underlying biochemical defects that lead to impaired mucilage release total 270
mucilage was extracted from seeds vigorously mixed using a ball mill (Voiniciuc et al 2015b 271
Voiniciuc and Guumlnl 2016) As previously described this intensive mechanical agitation 272
effectively removes all mucilage polysaccharides resulting in seeds that are no longer stained 273
by RR (Fig 4A) The monosaccharide composition of the total mucilage extracted from hydrated 274
seeds was quantified using high-performance anion-exchange chromatography with pulsed 275
amperometric detection (HPAEC-PAD Supplemental Table S2) Rha and GalA the building 276
blocks of the RG I backbone represent around 90 of total mucilage extracted from wild-type 277
Arabidopsis seeds (Fig 4 Voiniciuc et al 2015c) The muci70-1 and muci70-2 mutations 278
reduced the absolute levels of Rha and GalA in total mucilage extracts by more than 50 279
compared to the wild-type control (Fig 4B) The gaut11-3 single mutant reduced pectin content 280
by around 30 compared to the wild type (Fig 4B) similar to the gaut11-4 allele (Supplemental 281
Table S2) Interestingly the absolute abundance of the minor sugars in the total mucilage 282
extracts increased by more than 40 in the muci70 and gaut11 mutants compared to wild type 283
(Supplemental Table S2) This suggests that both MUCI70 and GAUT11 are particularly 284
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
important for the production and release of RG I but are not indispensable for the release of the 285
minor mucilage components For comparison a knockout insertion in the MYB5 transcription 286
factor which promotes seed coat differentiation and mucilage production (Li et al 2009 287
Voiniciuc et al 2015c) significantly decreased the content of all sugars found in total mucilage 288
extracts (Supplemental Table S2) Therefore muci70 and gaut11 mutants are deficient in the 289
production and release of pectic polysaccharides In contrast to the gaut11-3 and gaut11-4 290
mutants the gaut10-1 and gaut14-1 knockout mutants identified by genotyping (Supplemental 291
Table S3) reduced Rha and GalA levels by only 8 to 13 (Supplemental Table S2) Consistent 292
with their normal RR staining phenotypes (Supplemental Fig S2) gaut10-1 and gaut14-1 thus 293
had a relatively minor influence on mucilage production Indeed analysis of the muci70-1 294
gaut11-3 double revealed that MUCI70 together with GAUT11 accounted for the biosynthesis 295
and release of 88 of GalA-containing polymers in total seed mucilage extracts (Fig 4B) Two-296
factor ANOVA analysis of the HPAEC-PAD data (Supplemental Table S4) indicated that the 297
muci70-1 and gaut11-3 mutations had purely additive effects on GalA abundance Since the 298
muci70-1 gaut11-3 total mucilage extracts also contained 84 less Rha than the wild type the 299
mutated genes controlled the content of mucilage pectin in a non-redundant manner (Fig 4B) 300
Compared to the single mutants the muci70-1 gaut11-3 double mutant released even more 301
minor sugars in total mucilage extracts (Supplemental Table S2) Since the minor sugars are 302
primarily derived from hemicelluloses (Voiniciuc et al 2015a Voiniciuc et al 2015b) the 303
observed chemotype is consistent with the specific loss of pectin 304
Besides the drastic deficiency of RG I backbone sugars mutations in MUCI70 and GAUT11 305
significantly increased the absolute amounts of Gal Glc and Man in total mucilage extracts (Fig 306
4B Supplemental Table S2) but had distinct effects on the content of Ara and Xyl Based on 307
ANOVA the muci70-1 and gaut11-3 mutations had purely additive effects on the content of Gal 308
while the increases in Glc and Man content were higher than expected (Supplemental Table 309
S4) The two muci70 alleles significantly decreased Ara content (26 to 32) relative to the wild 310
type and gaut11 mutants ANOVA confirmed that only MUCI70 influenced the presence of Ara 311
(Supplemental Table S4) Surprisingly muci70 and gaut11 single mutants had polarizing effects 312
on Xyl content Relative to wild type muci70 single mutants increased Xyl abundance by 73 to 313
87 while the gaut11 single mutants and the muci70-1 gaut11-3 double mutant decreased Xyl 314
content by 43 to 47 (Supplemental Table S2) 315
To further investigate the structure of pectin and other polysaccharides glycosyl linkage 316
analysis was performed on total mucilage extracts (Table I) Relative to the wild-type control the 317
total mucilage extracts of both the muci70-1 and gaut11-3 mutants contained significant 318
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reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
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27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
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28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
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29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
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30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
10
important for the production and release of RG I but are not indispensable for the release of the 285
minor mucilage components For comparison a knockout insertion in the MYB5 transcription 286
factor which promotes seed coat differentiation and mucilage production (Li et al 2009 287
Voiniciuc et al 2015c) significantly decreased the content of all sugars found in total mucilage 288
extracts (Supplemental Table S2) Therefore muci70 and gaut11 mutants are deficient in the 289
production and release of pectic polysaccharides In contrast to the gaut11-3 and gaut11-4 290
mutants the gaut10-1 and gaut14-1 knockout mutants identified by genotyping (Supplemental 291
Table S3) reduced Rha and GalA levels by only 8 to 13 (Supplemental Table S2) Consistent 292
with their normal RR staining phenotypes (Supplemental Fig S2) gaut10-1 and gaut14-1 thus 293
had a relatively minor influence on mucilage production Indeed analysis of the muci70-1 294
gaut11-3 double revealed that MUCI70 together with GAUT11 accounted for the biosynthesis 295
and release of 88 of GalA-containing polymers in total seed mucilage extracts (Fig 4B) Two-296
factor ANOVA analysis of the HPAEC-PAD data (Supplemental Table S4) indicated that the 297
muci70-1 and gaut11-3 mutations had purely additive effects on GalA abundance Since the 298
muci70-1 gaut11-3 total mucilage extracts also contained 84 less Rha than the wild type the 299
mutated genes controlled the content of mucilage pectin in a non-redundant manner (Fig 4B) 300
Compared to the single mutants the muci70-1 gaut11-3 double mutant released even more 301
minor sugars in total mucilage extracts (Supplemental Table S2) Since the minor sugars are 302
primarily derived from hemicelluloses (Voiniciuc et al 2015a Voiniciuc et al 2015b) the 303
observed chemotype is consistent with the specific loss of pectin 304
Besides the drastic deficiency of RG I backbone sugars mutations in MUCI70 and GAUT11 305
significantly increased the absolute amounts of Gal Glc and Man in total mucilage extracts (Fig 306
4B Supplemental Table S2) but had distinct effects on the content of Ara and Xyl Based on 307
ANOVA the muci70-1 and gaut11-3 mutations had purely additive effects on the content of Gal 308
while the increases in Glc and Man content were higher than expected (Supplemental Table 309
S4) The two muci70 alleles significantly decreased Ara content (26 to 32) relative to the wild 310
type and gaut11 mutants ANOVA confirmed that only MUCI70 influenced the presence of Ara 311
(Supplemental Table S4) Surprisingly muci70 and gaut11 single mutants had polarizing effects 312
on Xyl content Relative to wild type muci70 single mutants increased Xyl abundance by 73 to 313
87 while the gaut11 single mutants and the muci70-1 gaut11-3 double mutant decreased Xyl 314
content by 43 to 47 (Supplemental Table S2) 315
To further investigate the structure of pectin and other polysaccharides glycosyl linkage 316
analysis was performed on total mucilage extracts (Table I) Relative to the wild-type control the 317
total mucilage extracts of both the muci70-1 and gaut11-3 mutants contained significant 318
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
11
reductions in 4-linked GalA the main building block of all pectin and 2-Rha characteristic of 319
unbranched RG I (Pettolino et al 2012 Voiniciuc et al 2015c) The abundance of 2-Rha and 320
4-GalA linkages was decreased by around 75 in muci70-1 and 25 in the gaut11-3 mutant 321
relative to wild type (Table I) consistent with impaired production of RG I and HG the two most 322
abundant pectic domains in seed mucilage (Voiniciuc et al 2015c) In contrast to their 323
consistent reduction of pectin linkages the muci70 and gaut11 mutants had distinct changes in 324
the abundance of minor mucilage components Only the muci70-1 mutant showed significant 325
decreases in both 3-Ara and 5-Ara (Table I) two linkages that could be derived from arabinan 326
side chains on RG I (Atmodjo et al 2013) Based on the ratio of 5-Ara to t-Ara linkages 327
arabinan chains in muci70-1 mucilage were estimated to be 30 shorter than in the wild type 328
While muci70-1 had a significant increase in the Xyl linkages previously associated with a highly 329
branched xylan polymer (Voiniciuc et al 2015a) gaut11-3 mucilage had significantly less xylan 330
(Table I) consistent with changes in Xyl detected with HPAEC-PAD (Fig 4B) The reduced 331
xylan content of the gaut11-3 mutant occurred with the presence of significantly more glycosyl 332
linkages associated with galactoglucomannan (t-Gal 4-Glc 4-Man and 46-Man) compared to 333
the wild type (Table I) To further investigate the distribution of polysaccharides we 334
immunolabeled whole seeds using the anti-mucilage CCRC-M30 and CCRC-M36 antibodies 335
and the anti-xylan CCRC-M139 antibody CCRC-M36 is specific for unbranched RG I (Ruprecht 336
et al 2017) while CCRC-M30 binds a yet-to-be-identified epitope unique to seed mucilage 337
(Pattathil et al 2010) All three antibodies labelled a uniform halo around wild-type seeds 338
(Supplemental Fig S3) In contrast muci70-1 seeds typically displayed only faint irregular 339
patches of CCRC-M36 and CCRC-M30 epitopes but more intense and broader labeling of 340
xylan (Supplemental Fig S3) Both the immunolabeling and glycosyl linkage data indicated that 341
mutations in MUCI70 resulted in a major decrease in RG I content accompanied by increased 342
xylan content in seed mucilage 343
We further validated that the muci70 defects resulted from the loss of a Golgi-localized 344
putative GT via the complementation of muci70 with a recombinant MUCI70 construct The 345
35SMUCI70-sYFP construct which was used to confirm the Golgi localization of the MUCI70 346
protein (Figs 1C to 1E) at least partially rescued the mucilage defects of the muci70-2 mutant 347
Multiple independent muci70-2 35SMUCI70-sYFP transformants produced seeds with uniform 348
RR-stained mucilage capsules (Fig 3I) and without the flotation defect that was frequently 349
observed for the muci70 mutant seeds (Fig 3C Fig 2F) The constitutive expression of 350
MUCI70-sYFP proteins tripled the RR-stained mucilage area of muci70-2 seeds hydrated in 351
water although this still fell short of the wild-type level (Fig 2E) In addition the 35SMUCI70-352
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12
sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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13
expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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14
6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
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693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
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stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
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24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
12
sYFP construct fully rescued the abundance of the Rha and GalA pectic sugars extracted from 353
muci70-2 mutant seeds (Fig 4B) but unexpectedly reduced the content of Xyl and Man relative 354
to the wild type Therefore the constitutive expression MUCI70 appeared to negatively affect 355
hemicellulose biosynthesis consistent with the significant increases of Xyl and Man in muci70 356
mutants 357
358
Unlike MUCI70 GAUT11 Functions as an HG α-14 GalA Transferase in vitro 359
As mentioned above GAUT11 belongs to the GAUT family of proven and putative HG α-14 360
GalA transferases (Sterling et al 2006 Atmodjo et al 2011 Biswal et al 2018) Since in 361
addition to RG I Arabidopsis seed mucilage is known to contain HG (Macquet et al 2007a 362
Voiniciuc et al 2013) particularly in the inner layer we tested whether GAUT11 had HG α-14 363
GalA transferase activity that could account for the mucilage defects observed in the gaut11 364
mutants A recombinant GAUT11∆39 protein containing N-terminal His8X and GFP tags instead 365
of the predicted transmembrane domain (Fig 1B) was expressed in the Human Embryonic 366
Kidney (HEK293) cell system (Moremen et al 2018) Purification of the expressed His8X
-GFP-367
GAUT11∆39 from the medium of the HEK293 cells followed by SDS-PAGE of the protein under 368
both reducing and non-reducing conditions (Fig 5A) revealed that GAUT11 does not form a 369
disulfide-linked dimeric or larger protein complex but rather exists primarily as a monomer in 370
vitro To determine if GAUT11 catalyzed HG elongation we tested whether the recombinant 371
protein incorporated radiolabeled GalA from UDP-[14C]GalA onto HG acceptors with degrees of 372
polymerization (DP) 7-23 (Fig 5B) Under these conditions measurable amounts of GalA[14C] 373
were detected in the product suggesting that GAUT11 is an HGGalA transferase Treatment of 374
the products with exopolygalacturonase (ExoPG) which specifically cleaves α-14 GalA 375
linkages confirmed that the products synthesized by GAUT11 were HG (Fig 5B) The 376
incorporation of GalA into HG by GAUT11 was linear over 45 minutes with a specific activity of 377
1473 plusmn 349 pmol GalA transferred min-1 mg-1 GAUT11 (Supplemental Fig S4A) To confirm that 378
HG was elongated and to identify the size of products formed GAUT11 was incubated with a 379
fluorescently-labeled HG acceptor of DP 13 (GalA13x-2AB) and UDP-GalA for three hours and 380
the products analysed by Matrix-Assisted Laser DesorptionIonization Time-of-Flight Mass 381
Spectrometry (MALDI-TOF MS) The resulting peak masses showed that GAUT11 catalyzed 382
the addition of up to six GalA residues or more onto the HG acceptor (Fig 5C) confirming that 383
GAUT11 is an HG α-14 GalA transferase Since putative GTs containing a DUF616 domain 384
have unknown biochemical functions (Fig 1A) we also tested whether MUCI70 had HGGalAT 385
activity A recombinant MUCI70∆77 protein without its transmembrane domain (Fig 1B) was 386
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13
expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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14
6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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15
35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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16
GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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17
accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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18
accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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19
production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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20
providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
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22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
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23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
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24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
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25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
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26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
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27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
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28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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expressed using the HEK293 cell system purified and assayed for HGGalA transferase 387
activity by MALDI-TOF MS No elongation of the GalA13X-2AB acceptor by MUCI70 was 388
observed (Supplemental Fig S4B) while under the same conditions GAUT11 exhibited 389
significant GalA13X-2AB acceptor elongation (Fig 5C) The results strongly suggest that reduced 390
synthesis of HG is the defect underlying the gaut11 mucilage phenotype In contrast MUCI70 391
lacks HGGalAT activity and exerts its effects via a different mechanism 392
393
Residual Mucilage Pectins in muci70 Mutant Require Xylan Produced by IRX14 394
GAUT11 and MUCI70 were both required for pectin synthesis in Arabidopsis seed mucilage 395
but they had contrasting effects on xylan abundance The constitutive expression of MUCI70-396
sYFP restored the mucilage RG I content to wild-type levels but reduced Xyl content while 397
mutations in MUCI70 elevated xylan production based on mucilage biochemical analysis and 398
immunolabeling (Fig 4 Supplemental Fig S3) These results prompted us to further investigate 399
the relationship between pectin and xylan production in SCE cells The irx14-1 mutant 400
previously shown to be essentially devoid of xylan (Voiniciuc et al 2015a) produced a normal 401
amount of pectin that detached from the seed surface following hydration in water (Figs 3G and 402
4C) We crossed the irx14-1 mutant to the muci70-1 mutant and isolated homozygous double 403
mutant plants by genotyping Relative to the single mutants the muci70-1 irx14-1 double mutant 404
showed more severe reductions than expected in both xylan and pectin-related sugars in total 405
mucilage extracts (Fig 4C) Data evaluation using ANOVA revealed that MUCI70 and IRX14 406
interact to control the abundance of most mucilage sugars (Supplemental Table S5) As a 407
notable exception only the muci70-1 mutation significantly altered the Ara content (Fig 4C) 408
which could be derived from arabinan 409
410
Cellulose Staining Reveals the Extent of Impaired Mucilage Release 411
To further investigate the underlying causes for the observed RR-staining defects (Fig 3) 412
seeds were stained with Pontamine Fast Scarlet S4B (abbreviated S4B) a cellulose-specific 413
fluorescent dye (Anderson et al 2010) and examined with confocal microscopy (Fig 6) The 414
distribution of cellulose stained with S4B around seeds hydrated in water provides a clear 415
overview of the primary cell wall and mucilage architecture Wild-type mucilage capsules 416
stained with S4B were characterized by long and regularly-spaced cellulosic rays (Fig 6A) 417
Although some muci70 and gaut11 seeds released mucilage after prolonged shaking in water 418
they showed altered distribution of cellulose compared to the wild type The muci70-1 and 419
muci70-2 seeds were surrounded by shorter rays which were curled rather than straight (Figs 420
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6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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18
accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
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693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
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stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
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36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
14
6B and 6C) The gaut11-3 and gaut11-4 mutants showed an intermediate defect with short but 421
relatively straight rays (Figs 6D and 6E) The curly ray phenotype of the muci70-2 mutant was 422
complemented by the constitutive expression of MUCI70s-YFP (Fig 6I) although the overall 423
intensity of S4B staining remained lower than the wild type Unlike either single mutant the 424
muci70-1 gaut11-3 double mutant displayed no S4B staining or only small patches around the 425
seed (Fig 6F) suggesting that most SCE cells did not release or produce mucilage While the 426
irx14-2 single mutant displayed clear S4B-labelled cellulosic regions (Fig 6G) despite the loss 427
of pectin adherence to the seed surface (Fig 3G) the muci70-1 irx14-2 double mutant was 428
essentially devoid of any S4B staining beyond the seed surface (Fig 6H) 429
430
MUCI70 and GAUT11 Are Essential for Mucilage Accumulation in Seeds 431
To further investigate if the observed RR staining defects (Fig 3) resulted from reduced 432
pectin biosynthesis rather than only poor extrusion in water dry seeds were pretreated with 433
ethylenediaminetetraacetic acid (EDTA) prior to water washes and RR staining Cation 434
chelators such as EDTA disrupt Ca2+-mediated pectic cross-links to promote mucilage release 435
from mutants that synthesize normal amounts of pectin but with a lower degree of 436
methylesterification (Rautengarten et al 2008 Voiniciuc et al 2013) Although the impaired 437
mucilage release defects of muci70 and gaut11 single mutants were partially supressed by the 438
EDTA pretreatment (Figs 7A to 7E) many muci70 seeds still floated on water (Figs 7B and 439
7C) and displayed the detachment of outer tangential primary cell walls as large sheets To 440
confirm that MUCI70 is indispensable for RG I biosynthesis we analyzed the composition of 441
total mucilage extracts (Fig 4A) following the EDTA pretreatment and of the remaining (de-442
mucilaged) seeds For the wild-type seeds the use of EDTA increased the relative proportion of 443
GalA and the absolute content of carbohydrates in total mucilage extracts (Fig 4D compare to 444
Figs 4B and 4C) Nevertheless the muci70-1 total mucilage extracts contained at least 53 445
less Rha and GalA than the wild type with the EDTA pretreatment (Fig 4D) or without it (Figs 446
4B and 4C) In contrast to the pectin-deficient total mucilage extracts the Rha and GalA content 447
of muci70-1 de-mucilaged seeds was similar to the wild type (Fig 4E) In addition the reduced 448
Ara content of muci70-1 total mucilage extracts was consistently detected with or without the 449
EDTA pretreatment (Figs 4B to 4D) Except for reduced Gal in the mucilage and remaining 450
seeds of muci70-1 following EDTA pretreatment the abundances of the other minor sugars 451
were not significantly different from those of the wild type (Figs 4D and 4E) Therefore the 452
EDTA pretreatment partially enhanced the extraction of pectic polysaccharides from seeds (Fig 453
7) but could not rescue the Rha and GalA deficiency of the muci70-1 mutant In addition the 454
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15
35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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16
GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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17
accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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18
accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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19
production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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20
providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
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25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
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27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
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28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
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29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
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- Figure 8
- Parsed Citations
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15
35SMUCI70-sYFP transgene complemented the defects of muci70-2 seeds pretreated with 455
EDTA (Fig 7I) including the aberrant primary cell wall detachment small RR-stained mucilage 456
capsules and seed flotation phenotypes Unlike the muci70 alleles the EDTA pretreatment 457
rescued the flotation phenotype (Figs 2F 3D and 3E) of gaut11-3 and gaut11-4 seeds (Figs 458
7D and 7E) Nevertheless both gaut11 mutants released mucilage capsules that were still 459
smaller than the wild type (Fig 7A) and surrounded by debris that may originate from the 460
primary cell wall (Figs 7D and 7E) 461
To investigate how the severe defects in pectin structure (Figs 3 6 7) affected the surface 462
morphology of SCE cells dry seeds were examined using scanning electron microscopy (SEM) 463
and wet seeds were examined with the transmitted light detector of a confocal microscope The 464
mutant seeds isolated in this study displayed wild-type surface area (Fig 2E) and overall seed 465
shape (Supplemental Fig S5) However close examination of SCE cells with SEM revealed 466
defective architecture of the primary and secondary cell walls in the RG I-deficient single and 467
double mutants examined (Fig 8) In the wild type cellulose-rich columellae are observed in the 468
center of every SCE cell (Fig 8A) and protrude like volcanoes from the surface of hydrated 469
seeds (Supplemental Fig S6A) The characteristic shape of the columellae is established by the 470
polar secretion of copious amounts of pectin early in seed coat development when mucilage is 471
produced (Young et al 2008) Mutations in RHM2MUM4 which supplies UDP-Rha for RG I 472
synthesis were previously shown to have flattened columellae as a result of reduced pectin 473
accumulation and smaller mucilage pockets (Usadel et al 2004 Western et al 2004) 474
Similarly the muci70 and to a lesser extent gaut11 mutants showed flatter columellae 475
compared to the wild type in transmitted light images of hydrated seeds (Supplemental Fig S6) 476
as well as in SEM micrographs of dry seeds (Fig 8) The impaired SCE cell surface morphology 477
of the muci70-2 mutant (Fig 8C) was fully rescued by the 35SMUCI70s-YFP transgene (Fig 478
8I) Consistent with their severe reductions in mucilage production (Fig 4) seeds of the muci70-479
1 gaut11-3 double mutant and the muci70-1 irx14-2 double mutant lacked detectable columellae 480
structures in both SEM (Figs 8F and 8H) and transmitted light images (Supplemental Figs S6F 481
and S6H) The SCE cells of the muci70-1 gaut11-3 double mutant in particular lacked the 482
hexagonal appearance of the wild type and were instead surrounded by radial primary walls 483
with highly irregular shapes (Fig 8F) Therefore the loss of both MUCI70 and GAUT11 484
completely flattened the landscape characteristic of the mucilage-secreting Arabidopsis seed 485
coat 486
487
DISCUSSION 488
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GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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18
accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
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production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
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providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
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composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
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22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
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23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
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24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
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25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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16
GTs Indispensable for Mucilage RG I Elongation Are Uncovered 489
Even though Arabidopsis seed mucilage consists primarily of unbranched RG I little to no 490
insight into its production has been gained in recent years While pectin production in SCE cells 491
remains enigmatic several studies in the last four years have characterized Arabidopsis seed 492
mucilage mutants that shed new light on the production of cellulose (Ben-Tov et al 2015 493
Griffiths et al 2015) xylan (Voiniciuc et al 2015a Hu et al 2016a Hu et al 2016b Ralet et 494
al 2016a) and galactoglucomannan (Yu et al 2014 Voiniciuc et al 2015b) Since cellulose 495
and hemicellulose represent relatively minor components of mucilage (Voiniciuc et al 2015c) 496
we hypothesized that screens for mucilage mutants have not been saturated and that novel 497
pectin-deficient mutants remained to be identified We therefore expanded the previously 498
described MUCI reverse genetic screen to systematically profile the expression of all 499
Arabidopsis CAZy genes during seed coat development This strategy identified MUCI70 a 500
member of a previously uncharacterized GT family as a promising candidate for mucilage 501
biosynthesis (Fig 1) Compared to the wild type two independent mutations in MUCI70 resulted 502
in seeds that released smaller mucilage capsules (Fig 2E) floated on water (Fig 2F) and 503
contained at least 60 less pectin in total mucilage extracts (Figs 4B and 4C) The reverse 504
genetic screen also yielded several GT8 family members (Supplemental Fig S1) including the 505
GATL5 and GAUT11 genes that were already linked to mucilage structure Although a gatl5 506
knockout mutant and a transgene complemented line have been analyzed in detail (Kong et al 507
2013) two gaut11 knockdown lines previously showed inconsistent mucilage phenotypes 508
(Caffall et al 2009) We therefore examined muci70 mutants alongside two novel gaut11-3 and 509
gaut11-4 alleles which showed similar defects in mucilage staining with RR (Fig 3) 510
Out of all the candidate genes screened MUCI70 and GAUT11 were found to be the most 511
important players for the biosynthesis and release of mucilage from seeds (Fig 2 Supplemental 512
Fig S2) The SCE cells of muci70 and gaut11 single mutants produced significantly less RG I 513
compared to the wild type based on their impaired mucilage staining phenotypes (Fig 3) their 514
Rha and GalA monosaccharide deficiency in total mucilage extracts (Fig 4B) and their glycosyl 515
linkage composition (Table I) Previously gaut11-2 non-adherent mucilage only appeared to 516
have decreased HG content but the content of Rha and uronic acids was determined via 517
separate techniques (Caffall et al 2009) By extracting the total mucilage polysaccharides (Fig 518
4A) and quantifying neutral and uronic sugars with a single HPAEC-PAD method (Voiniciuc and 519
Guumlnl 2016) we found that two independent mutations in GAUT11 showed significant 520
reductions in GalA as well as Rha monosaccharides which corresponded to lower amounts of 521
glycosyl linkages found in RG I and HG backbones (Table I) To rule out that mucilage 522
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17
accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
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18
accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
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36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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17
accumulated normally but was not effectively released upon hydration we pre-treated seeds 523
with EDTA a cation chelator capable of rescuing mucilage defects dependent on HG-calcium 524
cross-links (Rautengarten et al 2008 Voiniciuc et al 2013) While EDTA pretreatment 525
extracted more mucilage from muci70 and gaut11 seeds (Fig 7) than water alone (Fig 3) all of 526
the single mutants still displayed RR staining defects relative to the wild type Indeed muci70-1 527
total mucilage extracts contained less than half of the Rha and GalA found in the wild type with 528
(Fig 4D) or without the EDTA pretreatment (Figs 4B and 4C) In contrast after EDTA 529
pretreatment and total mucilage extraction wild-type and muci70-1 seeds contained similar 530
amounts of Rha and GalA (Fig 4E) Therefore MUCI70 was indispensable for the production of 531
RG I in SCE cells Both muci70 and gaut11 single mutants showed noticeably flatter columellae 532
in confocal images of hydrated seeds (Supplemental Fig S6) as well as SEM micrographs of 533
dry seeds (Fig 8) consistent with the accumulation of significantly less mucilage than in the 534
wild type In contrast to the major defects that resulted from the loss of either MUCI70 or 535
GAUT11 a gatl5 knockout mutant was previously reported to have wild-type mucilage 536
monosaccharide and glycosyl linkage composition (Kong et al 2013) Therefore we propose 537
that MUCI70 and GAUT11 are indispensable for the production of the majority of pectin in 538
Arabidopsis seed mucilage while GATL5 might only influence the final organization or 539
macromolecular size of these polymers 540
541
MUCI70 and GAUT11 Are Required for the Production of Distinct RG I Domains 542
Despite containing putative GT domains with distinct primary structures MUCI70 and 543
GAUT11 have similar protein topologies (Fig 1B) and transcriptional profiles in developing 544
seeds and embryos (Fig 2C) Insertions in either MUCI70 or GAUT11 significantly reduced the 545
content of RG I and HG-derived monosaccharides by around 60 and 30 respectively (Fig 546
4 Supplemental Table S2) The muci70-1 gaut11-3 double mutant nearly eliminated the 547
production of RG I in SCE cells as only 12 to 16 of the wild-type Rha and GalA sugars 548
remained (Fig 4B Supplemental Table S2) and seeds hydrated in EDTA or water released 549
little to no mucilage (Figs 3 5 6) ANOVA of the mucilage monosaccharide composition 550
indicated that the muci70-1 and gaut11-3 mutations had purely additive effects on GalA 551
abundance but partially overlapping effects on Rha content (Supplemental Table S4) 552
Furthermore while muci70 and gaut11 single mutants still displayed columellae albeit flatter 553
and wider than the wild type the muci70-1 gaut11-3 double mutant completely flattened the 554
surface of SCE cells (Supplemental Fig S6) and impaired the shape of their radial walls (Fig 555
8) The defects in seed surface morphology are consistent with severely impaired mucilage 556
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
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Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
18
accumulation in the SCE cells as previously reported for the pectin-deficient mum4 mutant 557
(Western et al 2004) and the myb5-1 transcription factor mutant (Li et al 2009) The pattern 558
of cellulose deposition in wild-type SCE cells is determined by the polarized secretion of copious 559
amounts of pectin into donut-shaped mucilage pockets (Voiniciuc et al 2015c) The resulting 560
volcano-shaped cytoplasmic columns are circled by cellulose synthases (Griffiths et al 2015) 561
leading to the deposition of cellulose-rich columellae (Mendu et al 2011) Therefore the 562
absence of cellulosic rays (Fig 6) and volcano-shaped collumellae (Fig 8 Supplemental Fig 563
S6) around muci70-1 gaut11-3 double mutant seeds likely resulted from reduced pectin 564
accumulation rather than direct changes in cellulose synthesis Overall the results suggest that 565
MUCI70 and GAUT11 are essential for the production of RG I domains whose structures or 566
biosynthesis are at least partially distinct but make up the bulk of Arabidopsis seed mucilage 567
In addition to their significant decreases in the glycosyl residues of the RG I backbone 568
muci70 and gaut11 mutants had distinct effects on Ara and Xyl two minor mucilage 569
components Besides Rha and GalA total mucilage extracts from both muci70 alleles were also 570
significantly deficient in Ara which corresponded to decreases in the arabinan side chain of RG 571
I (Table I) The 5-linked Ara content was reduced by 70 in the muci70-1 mutant compared to 572
the wild type (Table I) In contrast the gaut11 mutants had normal Ara content but a significant 573
decrease in Xyl (Fig 4B) derived from a highly branched xylan polymer found in wild-type total 574
mucilage extracts (Table I Voiniciuc et al 2015a) Although most of the RG I found in mucilage 575
released from mature seeds is unbranched (Voiniciuc et al 2015c) its backbone is likely 576
synthesized in a branched form in the Golgi apparatus and is subsequently modified in the 577
extracellular space Mutant seeds deficient in β-galactosidase (Dean et al 2007 Macquet et 578
al 2007b) or α-arabinofuranosidase activity (Arsovski et al 2009) contain more galactan or 579
arabinan RG I branches and display severely impaired mucilage release We therefore 580
hypothesize that MUCI70 and GAUT11 participate in the production of two distinct RG I 581
domains which contain arabinan and xylan side-chains respectively Mucilage was recently 582
demonstrated to contain xylan branches on RG I which mediate the adherence of pectin to 583
seeds (Ralet et al 2016b) 584
585
Novel Links between Pectin and Hemicellulose Biosynthesis 586
While the biological function of mucilage in Arabidopsis seeds remains unclear the 587
architecture of this gelatinous wall is primarily determined by the structure of RG I its major 588
component With the exception of upstream transcriptional regulators (Voiniciuc et al 2015c) 589
the mutants that display the most severe defects in mucilage release are directly involved the 590
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
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30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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19
production of nucleotide sugars for RG I biosynthesis or its metabolism in the wall (Usadel et al 591
2004 Dean et al 2007 Macquet et al 2007b Arsovski et al 2009) As discussed in the 592
preceding paragraph we found compelling evidence that MUCI70 and GAUT11 are required for 593
the synthesis and release of mucilage pectin By demonstrating that GAUT11 catalyzes HG 594
elongation in vitro (Fig 5 Supplemental Fig S4A) we propose that the synthesis of HG or of an 595
HG-glycan region is essential for mucilage RG I production However we cannot exclude the 596
alternative hypothesis that GAUT11 could utilize additional donor and acceptor substrates and 597
might therefore play a more direct role in RG I backbone elongation In contrast to GAUT11 598
MUCI70 purified from HEK293 cells did not appear to be involved in the elongation of HG 599
domains (Supplemental Fig S4B) The severe deficiency of RG I in muci70 total mucilage 600
extracts suggest that MUCI70 may be more directly involved in its synthesis So far the other 601
GTs known to be involved in the production of mucilage were found to only affect the structure 602
of a single class of polysaccharides pectin hemicellulose or cellulose For instance the irx14 603
mutant SCE cells had a nearly complete loss of xylan but did not significantly alter the content 604
of other mucilage polymers (Fig 4C Voiniciuc et al 2015a) In contrast mutations in MUCI70 605
andor GAUT11 reduced Rha and GalA content and significantly increased the absolute 606
amounts of Gal Glc and Man in mucilage extracts (Fig 4B) the building blocks of 607
galactoglucomannan (Table I) The greater abundance of minor sugars in total mucilage 608
extracts indicates that muci70 and gaut11 unlike the myb5-1 transcription factor mutant 609
(Supplemental Table S2) are not deficient in the release of all mucilage polymers but are 610
specifically involved in pectin production Relative to the wild type the gaut11-3 single mutant 611
contained a three-fold increase in the content of galactoglucomannan while the muci70-1 612
gaut11-3 double mutant had a four-fold increase (Supplemental Table S2) Since highly 613
branched galactoglucomannans have gelling properties akin to pectin and are known to control 614
the architecture of wild-type mucilage (Voiniciuc et al 2015b) a potential explanation for the 615
observed changes is that SCE cells may attempt to compensate for the reduced synthesis of 616
pectic domains by producing more hemicellulosic polymers with mucilaginous properties 617
In addition to the elevated content of galactoglucomannan-related sugars when RG I content 618
was reduced we discovered that xylan biosynthesis is indispensable for at least one RG I 619
domain Mutations in several GAUT genes were previously found to impair the production of 620
pectin as well as xylan (Orfila et al 2005 Pentildea et al 2007 Persson et al 2007 Caffall et al 621
2009) Although no requirement for xylan in pectin elongation was previously described there is 622
evidence that these two classes of polysaccharides can be covalently linked Proteoglycans that 623
contain both the pectins RG I and HG as well as xylan have been identified (Tan et al 2013) 624
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
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33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
20
providing an example of a polymer that could require an RG I domain as a possible primer for 625
synthesis of a xylan glycan Consistent with previous reports we found that gaut11 total 626
mucilage was deficient in both pectin and xylan To our initial surprise two independent muci70 627
mutants contained significantly more xylan than the wild type in the total mucilage extracts 628
despite a more severe reduction of RG I compared to gaut11 alleles (Fig 4 Supplemental 629
Table 2) These findings were supported by the more intense labeling of mucilage xylan by 630
CCRC-M139 and reduced detection of RG I with CCRC-M36 (Supplemental Fig S3) Although 631
irx14 mutants alone had no effect on pectin content in total mucilage extracts (Fig 4C Voiniciuc 632
et al 2015a Hu et al 2016) muci70 irx14 double mutant seeds were more deficient in RG I 633
than the muci70 single mutants (Fig 4C) ANOVA of monosaccharide composition indicated 634
that muci70 and irx14 mutations have synergistic effects on RG I production (Supplemental 635
Table S5) Since the muci70 irx14 seeds did not release any mucilage and showed only traces 636
of columellae (Figs 6 to 8 Supplemental Fig S6) the xylan-pectin connections were found to 637
be especially important for mucilage production in the muci70 background 638
639
Gaining Insight into the Biological and Biochemical Roles of DUF616 Proteins 640
An impasse in the biosynthesis of HG was solved 12 years ago by the first enzymatic 641
characterization of a GT involved in its elongation (Bacic 2006 Sterling et al 2006) However 642
the production of the RG I backbone the only polysaccharide in plants with a repeating 643
disaccharide backbone has remained a mystery since then In this study we identified MUCI70 644
as a putative GT from a novel CAZy family and demonstrated that it is indispensable for RG I 645
elongation in the Golgi apparatus of SCE cells and its release upon seed hydration We also 646
showed that GAUT11 has HG α-14 GalA transferase activity (Fig 5 Supplemental Fig S4A) 647
suggesting that the synthesis of HG may also be required for RG I elongation in mucilage The 648
enzymatic characterization of MUCI70 and functional analysis of other DUF616 proteins should 649
shed additional light on pectin biosynthesis Only one plant protein containing a DUF616 650
domain TOD1 has a known biochemical activity and functions as an alkaline ceramidase 651
involved in regulating turgor in guard cells and pollen tubes (Chen et al 2015) TOD1 appears 652
to be an anomaly among DUF616-containing proteins in Arabidopsis because it was an outlier 653
in our MUCI70 phylogenetic tree and lacks orthologs in early diverging land plants (Fig 1A) A 654
tod1 suppressor screen surprisingly identified that a mutation in GAUT13 which encodes a 655
putative pectin GT rescued the low seed set of the tod1 mutant (Chen et al 2015) Since a 656
gaut mutant was identified as a suppressor tod1 mutant pollen tubes were hypothesized to 657
contain more pectin which may reduce their growth potential Nevertheless the cell wall 658
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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21
composition of tod1 mutants was not tested so the link between pectin biosynthesis and 659
alkaline ceramidase activity is indirect and requires further investigation Based on the results 660
presented here MUCI70 is directly involved in pectin biosynthesis and thus likely has an activity 661
distinct from TOD1 662
Our characterization of muci70 and gaut11 single and double mutants indicates that 663
MUCI70 and GAUT11 are required for the synthesis of two distinct pectic regions associated 664
with RG I a view consistent with the latest model of pectin biosynthesis (Atmodjo et al 2013) 665
The additive effects of muci70-1 and gaut11-3 mutations on GalA levels suggests that MUCI70 666
and GAUT11 do not function in consecutive steps of pectin elongation Rather with the 667
demonstrated HG α-14 GalA transferase activity of GAUT11 the results suggest that GAUT11 668
synthesizes an HG region required for or associated with RG I Meanwhile MUCI70 could 669
potentially facilitate the transfer of Rha andor GalA or possibly arabinan or RG I 670
oligosaccharides into or onto RG I Although RG I is found in the walls of all growing plant cells 671
rhamnosyltransferases or galacturonosyltransferases involved RG I elongation have not yet 672
been identified Since MUCI70 is indispensable for the production of Arabidopsis seed 673
mucilage its biochemical activity should be comprehensively tested in future studies as should 674
the role of the GAUT11-synthesized HG glycan in mucilage RG I synthesis To accomplish this 675
will require technical advances in the purification of donor and acceptor substrates as well as 676
the establishment of robust in vitro assays for RG I biosynthesis Advancements in this area 677
have only recently emerged (Uehara et al 2017) and further developments should make it 678
feasible to determine if the promising candidates identified in this study can incorporate Rha 679
GalA or other carbohydrates into RG I 680
681
MATERIALS AND METHODS 682
Plant Material 683
The T-DNA insertion mutants analyzed in this study are listed in Supplemental Table S1 and 684
were selected from the SALK (Alonso et al 2003) and SAIL (Sessions et al 2002) collections 685
using the T-DNA Express tool (httpsignalsalkeducgi-bintdnaexpress) Mutant seeds and the 686
ST-RFP (N799376) marker were obtained from the Nottingham Arabidopsis Stock Centre 687
(NASC httparabidopsisinfo) Plants were grown in constant light as previously described 688
(Voiniciuc et al 2015c Voiniciuc et al 2015b) and seeds were harvested into separate bags 689
for each plant Mutants were genotyped by Touch-and-Go PCR (Berendzen et al 2005) 690
according to the SALK primer design tool (httpsignalsalkedutdnaprimers2html) The 691
primers are listed in Supplemental Table S3 692
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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22
693
In silico Analysis of Proteins 694
MUCI70-related protein sequences from three species and Arabidopsis (Arabidopsis thaliana) 695
GAUT sequences were obtained from Phytozome (Goodstein et al 2012) Phylogenetic 696
analysis was conducted using the MEGA60 software (Tamura et al 2013) as previously 697
described method (Hall 2013) Alignments were performed using the MUSCLE method and the 698
evolutionary history was inferred using the Maximum Likelihood method Trees were built using 699
the best model found including all sites (LG+G for MUCI70-related proteins LG+G+I for 700
GAUT family) Tree reliability was evaluated by the bootstrap method (500 replicates) The 701
topology of MUCI70 and GAUT11 proteins was assessed using the extended consensus TM 702
alpha helix prediction (AramTmMultiCon) tool in ARAMEMNON (Schwacke et al 2003) 703
704
RNA Isolation and RT-qPCR Analysis 705
Silique development was staged using non-toxic paint (Dean et al 2011) and three 7 DPA 706
siliques were harvested per plant (biological replicate) Silique RNA was isolated with the 707
RNeasy Plant Mini Kit (Qiagen) and was treated with DNase I as recommended by the 708
manufacturer For each biological replicate 200 ng of RNA was used as template for the iScript 709
cDNA Synthesis Kit (Bio-Rad) the expression of each gene was quantified at least twice using 710
iQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyiQ system Primers for transcript 711
quantification (Supplemental Table S3) were designed with Primer-BLAST (Ye et al 2012) or 712
QuantPrime (Arvidsson et al 2008) UBQ5 and elF4A1 served as reference genes (Gutierrez et 713
al 2008) and fold changes in target gene expression normalized to the geometric mean of the 714
two reference genes were calculated in Microsoft Excel according to a published method 715
(Fraga et al 2008) 716
717
Seed Mucilage Staining 718
Ruthenium red (RR VWR International GmbH Cat A34880001) staining of pectin was 719
performed as recently described (Voiniciuc et al 2015b Voiniciuc et al 2015a) using cell 720
culture plates with 24 wells (VWR International GmbH Cat 734-2325) The effect of cation 721
removal on mucilage release was tested by mixing seeds with water or 50 mM EDTA pH 95 for 722
60 min at 125 rpm before rinsing with water twice and staining with 001 (wv) RR All RR 723
images were acquired with a Leica DFC 295 camera equipped on a Leica MZ12 724
stereomicroscope and processed uniformly in Fiji (httpfijiscFiji Schindelin et al 2012) RR-725
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
23
stained mucilage and seed areas were quantified in Fiji using a semi-automated protocol 726
(Voiniciuc et al 2015b) 727
728
Mucilage cellulose staining was performed similarly to a published method (Voiniciuc et al 729
2015a) Seeds were first mixed with water in a 24-well plate on a horizontal shaker (15 min 100 730
rpm) After the water was removed cellulose was stained with 0025 (wv) S4B (now sold as 731
Direct Red 23 Sigma-Aldrich 212490-50G) in 50 mM NaCl solution (60 min 100 rpm) The dye 732
was then removed and the seeds were mixed with 500 microL water and transferred to glass slides 733
Optical sections were acquired with a Leica SP8 confocal system (552 nm excitation 600 to 650 734
nm emission) equipped with photomultipliers for fluorescence as well as transmitted light 735
736
Statistical Analyses 737
As previously described (Voiniciuc et al 2015a) significant changes relative to the wild type 738
were detected using the Students t-test (two-tailed distribution assuming equal variance of two 739
samples) The effects of two independent mutations on mucilage monosaccharide composition 740
were evaluated using two-factor ANOVA performed with the Real Statistics Resource Pack 741
(httpwwwreal-statisticscom) for Microsoft Excel 2010 742
743
Monosaccharide Composition of Total Mucilage Extracts 744
Total mucilage polysaccharides were extracted from 5 mg of seeds and analyzed as described 745
in a recent method (Voiniciuc and Guumlnl 2016) except that polymers were hydrolyzed for 90 min 746
at 120degC For each genotype the seeds of at least three different plants were examined as 747
independent biological replicates Monosaccharides were separated and quantified via HPAEC-748
PAD using a Dionex DX-600 system equipped with CarboPac PA20 guard and analytical 749
columns (Voiniciuc et al 2015b) For each dataset all genotypes were grown harvested 750
processed and analyzed simultaneously For the EDTA pretreatment 5 mg of dry seeds were 751
hydrated in 500 microL of 50 mM EDTA (pH 95) and then used for the total mucilage extraction 752
(Voiniciuc and Guumlnl 2016) Afterwards 300 microL of the supernatant was transferred to a 2 mL 753
screw-cap tube Polymers were precipitated by adding 1500 microL of absolute ethanol and vortex 754
mixing Following centrifugation (2 min at 20000 xg) the supernatant was discarded The 755
precipitated mucilage polymers were washed with 500 microL of 70 ethanol and then 756
resuspended in 300 microL of acetone before drying for 5 min at 60degC The seeds remaining from 757
the EDTA pretreatment and total mucilage extraction were washed twice with 1 mL of water 758
and ground using steel balls at 30 Hz for 130 min using a ball mill (Retsch MM400) De-759
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
24
mucilaged seed polysaccharides were washed twice with 70 ethanol once with 760
chloroformmethanol (11 vv) and once with acetone The insoluble polymers were then 761
resuspended in 300 microL of acetone and dried for 5 min at 60degC The monosaccharide 762
composition of total mucilage and de-mucilaged seeds after EDTA pretreatment was analyzed 763
as described above using Ribose as an internal standard 764
765
Whole Seed Immunolabeling 766
Monoclonal antibodies directed against xylan were obtained from CarboSource 767
(httpwwwccrcugaedu~carbosourceCSS_homehtml) Immunolabeling of seeds hydrated in 768
water was performed as previously described in detail (Voiniciuc et al 2015a Voiniciuc 2017) 769
using Alexa Fluor 488 goat anti-mouse secondary antibodies (Molecular Probes ThermoFisher 770
Scientific) Images of immunolabeled seeds (with or without counterstaining with S4B) were 771
acquired on a Leica SP8 confocal microscope using the following settings Alexa Fluor signal 772
(488 nm excitation 500-530 nm emission) and S4Bintrinsic seed fluorescence (552 nm 773
excitation 590-700 nm emission) The muci70-1 mutant was analyzed alongside previously 774
described wild-type seeds (Voiniciuc et al 2015a Voiniciuc 2017) 775
776
Glycosyl Linkage Analysis of Seed Mucilage 777
Glycosyl linkage analysis of total mucilage extracted with water from 60 mg of seeds was 778
performed as previously described (Voiniciuc et al 2015b Voiniciuc et al 2015a) For 779
genotype three plants (biological replicates) were analyzed in parallel After uronic acid 780
reduction (Gibeaut and Carpita 1991) extensive dialysis dimethyl sulfoxide solubilisation and 781
methylation (Gille et al 2009) the polysaccharides were hydrolyzed derivatized to the 782
corresponding alditol acetates and analyzed by GC-MS (Foster et al 2010) using sodium 783
borodeuteride for the reduction The glycosyl linkage composition was normalized to the 784
absolute abundance of each sugar residue quantified using HPAEC-PAD analysis of an aliquot 785
of the extracted mucilage Polysaccharide composition was calculated as described in a 786
detailed protocol (Pettolino et al 2012) with a minor modification (t-Xyl was assigned to xylan) 787
788
Scanning Electron Microscopy (SEM) 789
Mature dry Arabidopsis seeds were sputter coated with a gold layer (ca 5 nm thickness 60mA 790
current) using a Cressington Sputter Coater 208 HR integrated with thickness controller MTM-791
20 (Cressington Scientific Instruments Ltd Watford UK) Afterwards several seeds for each 792
genotype were mounted on a typical electron microscopy stub using a carbon adhesive tape 793
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
25
The SEM images were acquired using LEO (Zeiss) 1550 field emission SEM (ZeissLEO 794
Oberkochen Germany) with an in-lens or secondary electron detector at 5-15kV acceleration 795
voltage at the Helmholtz Nano Facility in Forschungszentrum Juumllich GmbH (Albrecht et al 796
2017) 797
798
Expression and Analysis of MUCI70-sYFP Proteins 799
The 35SMUCI70-sYFP construct was assembled using ligation-independent cloning (LIC) and 800
the pCV01 vector (Voiniciuc et al 2015b) Primers containing LIC adapters (Supplemental 801
Table S3) and Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used to 802
amplify a 3275 bp MUCI70 fragment (from ATG up to but excluding the stop codon) from 803
Arabidopsis genomic DNA The gel-purified MUCI70 PCR product was then used for LIC as 804
described (De Rybel et al 2011) The 35SMUCI70-sYFP plasmid was verified by Sanger 805
sequencing and introduced in Agrobacterium tumefaciens GV3101pMP90pSOUP cells 806
Arabidopsis plants were transformed using a modified floral spray method (Weigel and 807
Glazebrook 2006) with an infiltration medium containing 5 (wv) sucrose and 002 (vv) 808
Silwet L-77 T1 seedlings were selected with a 10 mgL glufosinate-ammonium spray (Sigma-809
Aldrich Cat 45520-100MG) 810
811
The subcellular localization of fluorescently-tagged proteins in stably transformed rosette leaf 812
epidermal cells was examined using a Leica SP8 microscope as previously described (Voiniciuc 813
et al 2015b) Plants expressing both MUCI70-sYFP and ST-RFP were obtained through 814
genetic crosses and fluorescent signals were sequentially acquired for each line scan sYFP 815
(488 nm excitation 505-550 emission) and RFP (552 nm excitation 590-635 nm emission) 816
817
Expression and Purification of GAUT11∆39 and MUCI70Δ77 in HEK293 Cells 818
Gateway expression vectors for transient expression in HEK293 cells and cloning and 819
expression methods were adapted from other publications (Moremen et al 2018) The 820
truncated coding sequences of GAUT11 and MUCI70 were PCR-amplified respectively from 821
TAIR clone U87017 (wwwarabidopsisorg) and from seven-day-old Arabidopsis whole seedling 822
cDNA (gift from Dr Melani Atmodjo University of Georgia) Specifically GAUT11 and MUCI70 823
were truncated to 3 beyond their predicted transmembrane domains ∆39 and ∆77 respectively 824
based on their Tm consensus from the Aramemnon database (Schwacke et al 2003 825
httparamemnonbotanikuni-koelnde) For the first PCR amplification the GAUT11∆39 F and 826
R primers and the MUCI70∆77 F and R primers were used to amplify the respective genes 827
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
26
(Supplemental Table S3) A second round of PCR amplification was performed using the attB F 828
and R universal primers (Supplemental Table S3) 829
830
The attB PCR products were cloned into the Gateway pDONR221 entry vector using the 831
Gateway BP Clonase II Enzyme (ThermoFisher) per the manufacturers instructions JM109 832
competent cells were transformed and plated on Luria-Bertani (LB) agar selection plates 833
containing 50 microgml kanamycin Colonies were selected and grown overnight at 37˚C at 250 834
rpm in 3 mL LB medium containing 50 microgml kanamycin Plasmids were isolated using the 835
GeneJet Plasmid Miniprep Kit (ThermoFisher) and sequence-confirmed (Macrogen) The 836
following primers were used for sequencing M13F M13R-pUC (Macrogen) and GAUT11 seq 837
or MUCI70 seq primers (Supplemental Table S3) The sequence-confirmed GAUT11 and 838
MUCI70 entry plasmids were cloned into the Gateway pGEn2-DEST Vector using the Gateway 839
LR Clonase II Enzyme (ThermoFisher) per the manufacturerrsquos instructions All steps were the 840
same as the BP Clonase II reaction except 100 microgml carbenicillin was used for selection The 841
following primers were used for sequencing pG2F pG2R and the GAUT11 seq or MUCI70 seq 842
primers (Supplemental Table S3) Glycerol stocks of sequence-confirmed colonies were stored 843
at -80˚C for future use 844
845
Sequence confirmed GAUT11∆39-pGEn2-DEST and MUCI70∆77-pGEn2-DEST cultures were 846
grown in 3 mL LB liquid medium containing 100 μgml carbenicillin at 250 rpm for 8 hours Two 847
mL of the culture was added to 500 mL of LB liquid medium with carbenicillin the culture 848
incubated at 37˚C and 250 rpm for 18 hours centrifuged at 4000 x g for 10 minutes at room 849
temperature and the supernatant discarded Plasmid isolation was performed using the 850
Invitrogen PureLink HiPure Plasmid Filter Maxiprep Kit (ThermoFisher) and the final DNA 851
concentration was measured using a NanoDrop spectrophotometer 852
853
Transfection of sterile GAUT11∆39 pGEn2-DEST or MUCI70∆77-pGEn2-DEST DNA into 854
HEK293 cells (Freestyle 293-F cells ThermoFisher) was done at a total concentration of 3 855
microgml total culture volume (250 mL for GAUT11 and 20 mL for MUCI70) with 9 microgml of 856
polyethyleneimine (linear 25 kDa PEI Polysciences) essentially as previously described 857
(Moremen et al 2018) A larger culture was required for GAUT11 due to lower purification 858
yields Following batch-mode production for 6 days the cells were separated from the medium 859
by centrifugation and the resulting clarified medium was filtered through a 045 micron nylon 860
filter GAUT11∆39 was purified using the AumlKTA FPLC system equipped with a 1 mL His-Trap 861
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
27
HP column (GE Healthcare) The column was equilibrated and washed with 50 mM HEPES pH 862
80 300 mM NaCl 10 mM imidazole and GAUT11∆39 was eluted at 1 mlmin with 50 mM 863
HEPES pH 80 300 mM NaCl using a gradient of 0-500 mM imidazole (20 column volumes) 864
MUCI70∆77 was purified using batch TALON metal affinity resin (Clontech) per the 865
manufacturerrsquos instructions using the same wash and elution buffers as for GAUT11∆39 866
Fractions containing the protein were exchanged into 50 mM HEPES pH 72 100 mM NaCl 867
and 15 glycerol storage buffer using a PD-10 column (GE Healthcare) The eluted proteins 868
were concentrated using a 30 kDa molecular weight cutoff Ultra Centrifugal Filter Unit (EMD 869
Millipore) and their concentrations measured by UV-Vis spectroscopy (Nanodrop) The resulting 870
purified GAUT11∆39 (17 mg) and MUCI70∆77 (13 mg) were distributed into 50 μL aliquots 871
flash frozen in liquid nitrogen and stored at -80˚C until use 872
Crude and purified protein preparations were separated by SDS-PAGE in the presence or 873
absence of reducing agent (25 mM DTT) and the proteins were visualized by staining of the gels 874
with Coomassie Brilliant Blue 875
876
Biochemical Analyses of His8X-GFP-GAUT11Δ39 and His8X-GFP-MUCI70Δ77 877
The radioactive GAUT11 α-14 GalA transferase (HGGalAT) activity assays (30 μL) contained 878
GAUT11 (200 nM 055 μg) 50 mM HEPES (pH 72) 025 (wv) BSA 025 mM MnCl2 10 microM 879
of a mixture of homogalacturonan acceptors with DP of 7-23 and 5 μM UDP-[14C]GalA For time 880
course assays an additional 95 μM of non-radiolabeled UDP-GalA was added (total of 100 microM 881
UDP-GalA) Reactions were incubated at 30degC and terminated by the addition of 5 μL of 400 882
mM NaOH For the GAUT11 HGGalAT time course the reactions were carried out from 0 to 883
240 min and terminated at the designated time points Product formation was measured using 884
the radioactive filter assay (Sterling et al 2005) 885
886
Sensitivity of HGGalAT reaction products to endopolygalacturonase (ExoPG) was measured as 887
follows HGGalAT reaction products produced in one-hour 30 μL reactions were mixed with 3 888
microL of 1M sodium acetate buffer pH 42 and 15 microL 2M acetic acid To half of the reaction tubes 889
4 U of purified Exo PG was added The reactions were incubated overnight at 30degC and 30 microL 890
of 1M NaOH was added to stop the reaction The final mixtures were assayed using the 891
radioactive filter assay Exo PG (EC 32167) was purified from Aspergillus tubengensis using 892
previously described methods (Kester et al 1996) except that a 5 mL HiTrap DEAE FF column 893
was used on the AumlKTA FPLC system (GE Healthcare) 894
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
28
895
UDP-D-[14C]GalpA was synthesized enzymatically from UDP-D-[14C]GlcpA (PerkinElmer) as 896
described (Liljebjelke et al 1995 Atmodjo et al 2011) The HG acceptor mix enriched for HG 897
oligosaccharides of DP 7-23 and the homogenous 13-mer GalA acceptor (GalA13X) were 898
generated by partial digestion of polygalacturonic acid with endopolygalacturonase and purified 899
by HPAEC-PAD as described (Doong and Mohnen 1998) 900
901
Analysis of HG-GalAT reaction products by MALDI-TOF MS was carried out as follows 902
HGGalAT reactions (20 μl) containing GAUT11 (1 μg) or MUCI70 (5 μg) 50 mM HEPES (pH 903
72) 005 (wv) BSA 025 mM MnCl2 100 μM GalA13X-2AB and 1 mM UDP-GalA were 904
analyzed using a Bruker LT mass spectrometer as previously described (Urbanowicz et al 905
2014) Aliquots (1 μl) of the reaction mixture were diluted with 10 μl of water and 1 μl was 906
spotted on the target plate containing air dried Nafion 117 solution (Sigma Jacobs and 907
Dahlman 2001) The samples were overlaid with 1 μL of matrix solution (20 mgml of 25-908
dihydroxbenzoic acid in aqueous 50 (vv) methanol) and the spot was crystallized under heat 909
The negative-ion spectra were recorded and at least 300 laser shots were summed to generate 910
each spectrum 911
912
The GalA13X-2AB acceptor was generated by labeling GalA13X with the fluorescent probe 2-913
aminobenzamide (2-AB) on the reducing end as described (Ishii 2002 Urbanowicz et al 914
2014) The sample was dialyzed four times against water in 3500 molecular weight cut-off 915
tubing (VWR Scientific) and recovered by lyophilization 916
917
Accession Numbers 918
Sequence data from this article can be found in the GenBankEMBL data libraries under 919
accession numbers listed in Supplemental Table S1 920
921
ACKNOWLEDGEMENTS 922
We thank Dr Rainer Schwacke (Forschungszentrum Juumllich) for helpful advice about the 923
MUCI70 and GAUT11 protein topology and the evolutionary history of DUF616 domains We 924
also thank Robert Amos for advice on GAUT11 cloning purification and optimization of the 925
MALDI and HGGalAT activity assays and Melani Atmodjo for preparation of the UDP-[14C]GalA 926
substrate Charles lsquoGrafrsquo Exum is thanked for assistance with the initial cloning of GAUT11 927
Gerardo Gutierrez-Sanchez is thanked for providing the Exo PG Aspergillus tubengensis fungal 928
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
29
stock 929
930
SUPPLEMENTAL MATERIAL 931
Supplemental Table S1 Mutants examined for mucilage defects 932
Supplemental Table S2 Monosaccharide composition of total mucilage extracted with water 933
Supplemental Table S3 Primer sequences used for genotyping RT-qPCR and cloning 934
Supplemental Table S4 ANOVA tables to test if MUCI70 and GAUT11 interact 935
Supplemental Table S5 ANOVA tables to test if MUCI70 and IRX14 interact 936
Supplemental Figure S1 Multiple GAUT genes are expressed in the seed coat 937
Supplemental Figure S2 RR staining of mucilage capsules around gaut mutant seeds 938
Supplemental Figure S3 Polysaccharide immunolabeling in seed mucilage capsules 939
Supplemental Figure S4 Biochemical analyses of GAUT11 and MUCI70 940
Supplemental Figure S5 Whole seed morphology visualized with SEM 941
Supplemental Figure S6 Protrusion of columellae from hydrated seeds 942
943
944
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
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- Figure 7
- Figure 8
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30
TABLES 945
Table I Glycosyl linkages in total mucilage extracted with water 946
Linkage abundance was normalized to the absolute monosaccharide levels (microg mg seed) of 947 the same mucilage extracts Data show the mean plusmn SD of three biological replicates per 948 genotype Bold values are significantly different from the wild type (Studentrsquos t test P lt 005) 949
Linkage Wild Type muci70-1 gaut11-3
Rhamnose
t-Rha 0113 plusmn 0000 0175 plusmn 0069 0204 plusmn 0093
2-Rha 9082 plusmn 0048 1949 plusmn 0141 6277 plusmn 0319
23-Rha 0220 plusmn 0025 0066 plusmn 0013 0156 plusmn 0031
24-Rha 0093 plusmn 0065 0051 plusmn 0010 0095 plusmn 0007
Arabinose
t-Ara 0056 plusmn 0007 0030 plusmn 0009 0042 plusmn 0014
5-Ara 0069 plusmn 0000 0021 plusmn 0008 0047 plusmn 0009
3-Ara 0167 plusmn 0012 0114 plusmn 0018 0208 plusmn 0021
Galactose
t-Gal 0153 plusmn 0012 0288 plusmn 0071 0313 plusmn 0034
2-Gal 0072 plusmn 0016 0127 plusmn 0010 0270 plusmn 0024
4-Gal 0097 plusmn 0001 0052 plusmn 0007 0108 plusmn 0008
6-Gal 0024 plusmn 0002 0021 plusmn 0007 0035 plusmn 0006
24-Gal 0036 plusmn 0008 0026 plusmn 0003 0045 plusmn 0004
46-Gal 0092 plusmn 0001 0075 plusmn 0026 0168 plusmn 0009
Glucose
t-Glc 0013 plusmn 0009 0014 plusmn 0007 0029 plusmn 0009
4-Glc 0246 plusmn 0037 0373 plusmn 0052 0767 plusmn 0089
34-Glc 0014 plusmn 0003 0014 plusmn 0003 0036 plusmn 0010
46-Glc 0029 plusmn 0003 0037 plusmn 0013 0101 plusmn 0025
Xylose
t-Xyl 0172 plusmn 0005 0335 plusmn 0062 0108 plusmn 0014
4-Xyl 0640 plusmn 0034 1070 plusmn 0090 0361 plusmn 0009
24-Xyl 0258 plusmn 0018 0372 plusmn 0026 0125 plusmn 0013
Mannose
4-Man 0061 plusmn 0002 0092 plusmn 0012 0157 plusmn 0013
46-Man 0159 plusmn 0018 0228 plusmn 0044 0562 plusmn 0041
Galacturonic Acid
t-GalA 0075 plusmn 0002 0049 plusmn 0007 0096 plusmn 0028
4-GalA 12175 plusmn 0647 3793 plusmn 0394 9252 plusmn 0625
24-GalA 0128 plusmn 0003 0060 plusmn 0018 0085 plusmn 0007
46-GalA 0165 plusmn 0001 0053 plusmn 0007 0146 plusmn 0033
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
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31
950 FIGURE LEGENDS 951
Figure 1 MUCI70 is a DUF616 protein related to glycosyltransferases 952
(A) Phylogenetic analysis of DUF616 proteins in Arabidopsis P patens and S moellendorffii 953 (B) Schematic of conserved domains in MUCI70 and GAUT11 proteins T transmembrane 954 domain (CndashE) Co-localization of MUCI70-sYFP with the Golgi marker ST-RFP (Teh and Moore 955 2007) in stably transformed Arabidopsis rosette leaf epidermal cells Scale bars = 50 aa (B) 10 956 microm (CndashE) 957 958 Figure 2 Analysis of T-DNA insertions in MUCI70 and GAUT genes 959
(A) Position of T-DNA insertions in MUCI70 and GAUT11 genes Ovals represent exons 960 connecting lines show introns outer lines depict untranslated regions Small arrowheads 961 indicate positions of RT-qPCR primers (B) Gene expression in wild-type (WT) siliques at three 962 different stages (DPA days post-anthesis two biological replicates per time point) (C) ATH1 963 GeneChip expression level (mean + SD) in general seed coats and embryos at Heart (H) 964 Linear Cotyledon (LC) and Maturation Green (MG) stages Data obtained by (Belmonte et al 965 2013) was extracted from the eFP Browser (Winter et al 2007) (D) Effects of T-DNA insertions 966 on MUCI70 and GAUT11 transcript abundance in whole siliques at 7 DPA In (B) and (D) data 967 show means + SD of two technical (B) or biological (D) replicates normalized to the geometric 968 mean of the UBQ5 and elfF4A1 reference genes and the relative expression of the first sample 969 was set as 10 in each series Scale bars = 250 bp (A and B) (E) Dimensions of RR-stained 970 mucilage capsules released from seeds in water Data show means + SD of five biological 971 replicates (gt20 seeds each) The 35SMUCI70-sYFP transgene partially rescued the mucilage 972 defect of the muci70-2 mutant (F) Percentage of seeds that float on water Data show means + 973 SD of 3 biological replicates (gt35 seeds each) 974 975 Figure 3 RR staining of mucilage polysaccharides around seeds hydrated in water 976
(AndashI) RR staining of mucilage released from seeds Stars mark seeds that float on water 977 Relative to wild-type seeds (A) muci70 and gaut11 single mutants release less mucilage (BndashE) 978 No mucilage is released from the muci70 gaut11 double mutant (F) or muci70 irx14 seeds (H) 979 In the irx14 single mutant (G) mucilage is released but detaches from the seed surface (I) The 980 35SMUCI70-sYFP transgene rescues the impaired mucilage release and the seed flotation 981 defects of the muci70-2 mutant Scale bars = 04 mm 982 983 Figure 4 Carbohydrate analysis of total mucilage extracted with water 984
(A) Overview of the total mucilage extraction which removes all polysaccharides from the seed 985 surface that can be stained with RR (B) and (C) Monosaccharide composition of total mucilage 986 extracted from seeds Data show mean + SD of four biological replicates per genotype 987 Significant changes from the wild type and between mutants are indicated by different red 988 letters (Studentrsquos t test P lt 005) The monosaccharide composition of the lines shown in Fig 989 4B is provided in Supplemental Table S2 along with the data for gaut11-4 gaut10-1 and 990 gaut14-1 mutants (D) Monosaccharide composition of the alcohol-insoluble residue (AIR) 991 isolated from total mucilage extracts following EDTA pretreatment and the remaining seeds 992 Data show mean + SD of three biological replicates Asterisks indicate a significant change 993 relative to the wild type (Studentrsquos t test P lt 005) 994 995 996 Figure 5 Purification and enzymatic characterization of His8X-GFP-GAUT11∆39 997
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
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- Parsed Citations
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32
(A) Coomassie stained SDS-PAGE of protein standard (S) 40 microL of combined medium and 998
HEK293 cells expressing His8X-GFP-GAUT11∆39 (lane 1) 40 microL of medium only from HEK293 999
cells expressing His8X-GFP-GAUT11∆39 (lane 2) 5 microg of purified recombinant protein under 1000
reducing (lane 3) and non-reducing conditions (lane 4) The expected molecular weight of His8X-1001
GFP-GAUT11∆39 is 911 kDa (B) Incorporation of [14C]GalA by His8X-GFP-GAUT11∆39 into 1002
products sensitive to exopolygalacturonase (Exo PG) The purified protein HG oligosaccharides 1003
(DP 6-23) and UDP-[14C]GalA were incubated for one hour An aliquot of the products was 1004
treated with (+) or without (-) Exo PG for 18 hours Data show mean + SE of two independent 1005
assays each with duplicate samples Exo PG treatment significantly degraded the product (P lt 1006
0001) based on ANOVA followed by Tukeyrsquos HSD test (C) MALDI-TOF MS of the products 1007
resulting from the incubation of His8X-GFP-GAUT11∆39 GalA13x-2AB acceptor and UDP-GalA 1008
for zero (upper panel) and three hours (lower panel) The mass differences between each peak 1009
are consistent with sequential addition of one GalA residue (176 Da) for each catalytic transfer 1010
Spectra are representative of two independent assays 1011
Figure 6 S4B staining of cellulose in mucilage capsules of seeds hydrated in water 1012
(AndashI) Single optical sections of fluorescent signals detected with confocal microscope Arrows 1013 show well-defined cellulosic rays (A and I) Asterisks indicate short curly rays observed in 1014 mutants with muci70 insertions No straight rays are observed in (FndashH) Scale bars = 150 microm 1015 1016 Figure 7 RR staining of mucilage polysaccharides around seeds hydrated in EDTA 1017
(AndashI) RR staining of seeds after EDTA pretreatment Arrows indicate detached sheets from the 1018 seed surface Stars mark floating seeds Scale bars = 040 mm 1019 1020 Figure 8 Surface morphology of Arabidopsis seed coat epidermal cells 1021
(AndashI) Scanning electron micrographs of mature dry seeds The letter ldquocrdquo marks the center of 1022 volcano-shaped columellae which are not detected in (F) Asterisks mark small remnants of 1023 columellae in (H) White dashed lines highlight the size of columellae while black dashed lines 1024 highlight primary walls surrounding epidermal cells Scale bars = 20 microm (AndashI) 1025 1026 1027
LITERATURE CITED 1028
Albrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res 1029 Facil JLSRF 3 A112 1030
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 1031 Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of 1032 Arabidopsis thaliana Science (80- ) 301 653ndash657 1033
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose 1034 reorientation during cell wall expansion in Arabidopsis roots Plant Physiol 152 787ndash96 1035
Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 1036 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic 1037 arabinan modification in Arabidopsis mucilage secretory cells Plant Physiol 150 1219ndash1038 1234 1039
Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a 1040
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
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37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
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- Parsed Citations
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33
flexible tool for reliable high-throughput primer design for quantitative PCR BMC 1041 Bioinformatics 9 465 1042
Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant 1043 Biol 64 747ndash779 1044
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller 1045 H V Mohnen D (2011) Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a 1046 plant cell wall pectin biosynthetic homogalacturonangalacturonosyltransferase complex 1047 Proc Natl Acad Sci U S A 108 20225ndash30 1048
Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash1049 5640 1050
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei 1051 J Harada CM Munoz MD et al (2013) Comprehensive developmental profiles of gene 1052 activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A 110 1053 E435-44 1054
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M 1055 Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE 2 a member of the GPI-anchored 1056 COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage 1057 secretory cells Plant Physiol 167 711ndash24 1058
Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE 1059 Ulker B (2005) A rapid and versatile combined DNARNA extraction protocol and its 1060 application to the analysis of a novel DNA marker set polymorphic between Arabidopsis 1061 thaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4 1062
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM 1063 Zhang J-Y et al (2018) Sugar release and growth of biofuel crops are improved by 1064 downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067 1065
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in 1066 Arabidopsis involved in secondary cell wall formation using expression profiling and 1067 reverse genetics Plant Cell 17 2281ndash95 1068
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA 1069 mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and 1070 seed testa Mol Plant 2 1000ndash14 1071
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase 1072 TOD1 is a key turgor pressure regulator in plant cells Nat Commun 6 1ndash10 1073
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall 1074 structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476 1075
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R 1076 Haughn GW (2011) Analysis of gene expression patterns during seed coat development in 1077 Arabidopsis Mol Plant 4 1074ndash91 1078
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC 1079 McCann MC Mansfield SD et al (2007) The Arabidopsis MUM2 gene encodes a beta-1080 galactosidase required for the production of seed coat mucilage with correct hydration 1081 properties Plant Cell 19 4007ndash4021 1082
Doong R Lou Mohnen D (1998) Solubilization and characterization of a 1083
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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34
galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan 1084 Plant J 13 363ndash374 1085
Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H 1086 Ulvskov P Geshi N (2006) Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-1087 localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic 1088 rhamnogalacturonan-II Plant Cell 18 2593ndash607 1089
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell 1090 Walls (Lignocellulosic biomass) Part II Carbohydrates J Vis Exp 37 e1745 1091
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr 1092 Protoc Essent Lab Tech First Edit John Wiley amp Sons Inc Hoboken pp 1ndash33 1093
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plants selective 1094 turnover and alteration of soluble and cell wall polysaccharides in grasses Plant Physiol 1095 97 551ndash561 1096
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by 1097 means of a forward chemical genetic approach using hydrolases Proc Natl Acad Sci U S A 1098 106 14699ndash704 1099
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W 1100 Hellsten U Putnam N et al (2012) Phytozome a comparative platform for green plant 1101 genomics Nucleic Acids Res 40 D1178ndashD1186 1102
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH 1103 Shawn DM Debolt S et al (2015) Unidirectional Movement of Cellulose Synthase 1104 Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in 1105 Mucilage Extrusion Adherence and Ray Formation Plant Physiol 168 502ndash520 1106
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz 1107 T Guerineau F Bellini C et al (2008) The lack of a systematic validation of reference 1108 genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction 1109 (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618 1110
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1111 1229ndash1235 1112
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying 1113 Arabidopsis irregular xylem mutants with pleiotropic phenotypes Crit Rev Biochem Mol 1114 Biol 9238 1ndash30 1115
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 1116 384ndash95 1117
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall 1118 that Can be Used as a Model for Genetic Analysis of Plant Cell Wall Structure and 1119 Function Front Plant Sci 3 64 1120
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W 1121 Zimmermann P (2008) Genevestigator V3 A Reference Expression Database for the 1122 Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5 1123
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan 1124 synthesized by Irregular Xylem 14 ( IRX14 ) maintains the structure of seed coat mucilage 1125 in Arabidopsis J Exp Bot 67 1243ndash1257 1126
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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35
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 1127 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis Plant Mol Biol 92 1128 25ndash38 1129
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly 1130 acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410 1131
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal 1132 AJ Jensen NB Soslashrensen C et al (2008) Identification of a xylogalacturonan 1133 xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302 1134
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and 1135 characterization of an exopolygalacturonase from Aspergillus tubingensis Eur J Biochem 1136 240 738ndash746 1137
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker 1138 L Mohnen D Western T et al (2013) GALACTURONOSYLTRANSFERASE-LIKE5 is 1139 involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17 1140
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) 1141 The Arabidopsis MYB5 transcription factor regulates mucilage synthesis seed coat 1142 development and trichome morphogenesis Plant Cell 21 72ndash89 1143
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and 1144 purification of uridine diphosphate [14C]galacturonic acid a substrate for pectin 1145 biosynthesis Anal Biochem 225 296ndash304 1146
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A 1147 Andersen MCF Clausen MH Scheller H V Jennifer A et al (2012) Pectin biosynthesis 1148 GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 1149 24 5024ndash36 1150
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The 1151 carbohydrate-active enzymes database (CAZy) in 2013 Nucleic Acids Res 42 490ndash495 1152
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical 1153 and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage 1154 Plant Cell Physiol 48 984ndash99 1155
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM 1156 (2007b) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-1157 galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed 1158 mucilage Plant Cell 19 3990ndash4006 1159
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive 1160 food polysaccharide Trends Food Sci Technol 24 64ndash73 1161
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S 1162 (2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates 1163 secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453 1164
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao 1165 Z Chapla D et al (2018) Expression system for structural and functional studies of human 1166 glycosylation enzymes Nat Chem Biol 14 156ndash162 1167
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J 1168 Biol Macromol 51 681ndash689 1169
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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36
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine 1170 BLAST and co-expression analyses Nucleic Acids Res 36 W320-6 1171
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by 1172 stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides 1173 consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 66 1301ndash1174 13 1175
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley 1176 KS Dupree P (2012) Putative glycosyltransferases and other plant Golgi apparatus 1177 proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51 1178
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein 1179 quantification for plant Golgi protein localisation and abundance Plant Physiol 166 1033ndash1180 43 1181
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 1182 2014 Evaluation of gene coexpression in agriculturally important plants Plant Cell Physiol 1183 55 1ndash7 1184
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP 1185 Scheller HV (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana 1186 inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 1187 613ndash622 1188
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM 1189 Morrison S McInerney P Hadi MZ et al (2012) Isolation and proteomic characterization 1190 of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall 1191 biosynthesis Plant Physiol 159 12ndash26 1192
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A 1193 Davis RH Chennareddy C et al (2010) A comprehensive toolkit of plant cell wall glycan-1194 directed monoclonal antibodies Plant Physiol 153 514ndash25 1195
Pentildea MJ Zhong R Zhou G-K Richardson EA OrsquoNeill MA Darvill AG York WS Ye Z-H 1196 (2007) Arabidopsis irregular xylem8 and irregular xylem9 implications for the complexity of 1197 glucuronoxylan biosynthesis Plant Cell 19 549ndash63 1198
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen 1199 D Somerville CR (2007) The Arabidopsis irregular xylem8 mutant is deficient in 1200 glucuronoxylan and homogalacturonan which are essential for secondary cell wall 1201 integrity Plant Cell 19 237ndash55 1202
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required 1203 for cellulose synthesis by regression analysis of public microarray data sets Proc Natl 1204 Acad Sci U S A 102 8633ndash8638 1205
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide 1206 composition of plant cell walls Nat Protoc 7 1590ndash607 1207
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L 1208 North HM (2016a) The affinity of xylan branches on rhamnogalacturonan I for cellulose 1209 provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat 1210 Plant Physiol pp002112016 1211
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L 1212 North HM Creacutepeau M-J et al (2016b) Xylans Provide the Structural Driving Force for 1213
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
37
Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178 1214
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A 1215 subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats 1216 Plant J 54 466ndash80 1217
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T 1218 Knox JP Hahn MG Clausen MH et al (2017) A Synthetic Glycan Microarray Enables 1219 Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1220 1104 1221
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL 1222 Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional 1223 studies in plants Plant Physiol 156 1292ndash9 1224
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S 1225 Rueden C Saalfeld S Schmid B et al (2012) Fiji an open-source platform for biological-1226 image analysis Nat Methods 9 676ndash682 1227
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer 1228 WB Fluumlgge U-I Kunze R (2003) ARAMEMNON a novel database for Arabidopsis 1229 integral membrane proteins Plant Physiol 131 16ndash26 1230
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics 1231 System Plant Cell 14 2985ndash2994 1232
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D 1233 (2006) Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan 1234 galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241 1235
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for 1236 measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 1237 343 231ndash236 1238
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular 1239 evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1240
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller 1241 JS et al (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan 1242 covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87 1243
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in 1244 polarized plant cells Nature 448 493ndash496 1245
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T 1246 Fujiyama K et al (2017) Biochemical characterization of rhamnosyltransferase involved in 1247 biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem Biophys Res 1248 Commun 486 130ndash136 1249
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis 1250 proteins synthesize acetylated xylan in vitro Plant J 80 197ndash206 1251
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in 1252 mucilage pectin synthesis and is required for the development of the seed coat in 1253 Arabidopsis Plant Physiol 134 286ndash295 1254
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-1255
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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38
protocol 7 e2323 1256
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western 1257 TL Estelle M Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3 1258 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed 1259 mucilage Plant Cell 25 944ndash59 1260
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from 1261 Arabidopsis Seeds Bio-protocol 6 e1801 1262
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by 1263 IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis 1264 Seeds Plant Physiol 169 2481ndash95 1265
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell 1266 Wall Plant Physiol 176 2590ndash2600 1267
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel 1268 B Guumlnl M (2015b) MUCILAGE-RELATED10 Produces Galactoglucomannan That 1269 Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 1270 169 403ndash420 1271
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How 1272 Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls Int J 1273 Mol Sci 16 3452ndash3473 1274
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) 1275 Extensive Natural Variation in Arabidopsis Seed Mucilage Structure Front Plant Sci 7 803 1276
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative 1277 rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of 1278 bioinformatics approaches PLoS One 7 e51129 1279
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are 1280 Inherent to Never-Dried Arabidopsis Primary Cell Walls Evidence from Solid-State Nuclear 1281 Magnetic Resonance Plant Physiol 168 871ndash884 1282
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios 1283 C Kazi F Lopes CT et al (2010) The GeneMANIA prediction server biological network 1284 integration for gene prioritization and predicting gene function Nucleic Acids Res 38 1285 W214-20 1286
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 1287 pdbprot4668 1288
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-1289 MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated 1290 by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the Arabidopsis 1291 Seed Coat Plant Physiol 134 296ndash306 1292
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An ldquoelectronic 1293 fluorescent pictographrdquo Browser for exploring and analyzing large-scale biological data 1294 sets PLoS One 2 e718 1295
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST 1296 A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 1297 13 134 1298
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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39
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis 1299 of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-1300 rich mucilage Plant Cell 20 1623ndash38 1301
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a 1302 Glucomannan Synthase is Involved in Maintaining Adherent Mucilage Structure in 1303 Arabidopsis Seed Plant Physiol 164 1842ndash1856 1304
1305
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
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- Figure 6
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- Figure 8
- Parsed Citations
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
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- Figure 6
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- Figure 8
- Parsed Citations
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
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Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
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Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
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Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
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Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
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Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
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Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
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Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
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Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
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Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
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Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
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Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
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Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
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Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
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Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
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Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
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Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
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Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
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Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
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Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
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De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
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Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
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Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
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Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
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Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
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Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science (80- ) 301 653ndash657
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Anderson CT Carroll A Akhmetova L Somerville CR (2010) Real-time imaging of cellulose reorientation during cell wall expansion inArabidopsis roots Plant Physiol 152 787ndash96
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Arvidsson S Kwasniewski M Riantildeo-Pachoacuten DM Mueller-Roeber B (2008) QuantPrime--a flexible tool for reliable high-throughputprimer design for quantitative PCR BMC Bioinformatics 9 465
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Atmodjo MA Sakuragi Y Zhu X Burrell AJ Mohanty SS Atwood JA Orlando R Scheller H V Mohnen D (2011)Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetichomogalacturonangalacturonosyltransferase complex Proc Natl Acad Sci U S A 108 20225ndash30
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Belmonte MF Kirkbride RC Stone SL Pelletier JM Bui AQ Yeung EC Hashimoto M Fei J Harada CM Munoz MD et al (2013)Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed Proc Natl Acad Sci U S A110 E435-44
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Berendzen K Searle I Ravenscroft D Koncz C Batschauer A Coupland G Somssich IE Ulker B (2005) A rapid and versatilecombined DNARNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsisthaliana ecotypes Col-0 and Landsberg erecta Plant Methods 1 4
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Cao Y Xiang D Provart NJ Ramsay L Ahad A White R Selvaraj G Datla R Haughn GW (2011) Analysis of gene expressionpatterns during seed coat development in Arabidopsis Mol Plant 4 1074ndash91
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Doong R Lou Mohnen D (1998) Solubilization and characterization of a galacturonosyltransferase that synthesizes the pecticpolysaccharide homogalacturonan Plant J 13 363ndash374
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibeaut DM Carpita NC (1991) Tracing cell wall biogenesis in intact cells and plantsthinsp selective turnover and alteration of soluble andcell wall polysaccharides in grasses Plant Physiol 97 551ndash561
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
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Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
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- Parsed Citations
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Parsed CitationsAlbrecht W Moers J Hermanns B (2017) HNF - Helmholtz Nano Facility J large-scale Res Facil JLSRF 3 A112
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Arsovski AA Popma TM Haughn GW Carpita NC Mccann MC Western TL (2009) AtBXL1 encodes a bifunctional beta-D-xylosidasealpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells PlantPhysiol 150 1219ndash1234
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Atmodjo MA Hao Z Mohnen D (2013) Evolving Views of Pectin Biosynthesis Annu Rev Plant Biol 64 747ndash779Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Bacic A (2006) Breaking an impasse in pectin biosynthesis Proc Natl Acad Sci 103 5639ndash5640Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ben-Tov D Abraham Y Stav S Thompson K Loraine A Elbaum R De Souza A Pauly M Kieber JJ Harpaz-Saad S (2015) COBRA-LIKE2 a member of the GPI-anchored COBRA-LIKE family plays a role in cellulose deposition in Arabidopsis seed coat mucilage secretorycells Plant Physiol 167 711ndash24
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Biswal AK Atmodjo MA Li M Baxter HL Yoo CG Pu Y Lee Y-C Mazarei M Black IM Zhang J-Y et al (2018) Sugar release and growthof biofuel crops are improved by downregulation of pectin biosynthesis Nat Biotechnol doi 101038nbt4067
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Brown DM Zeef LAH Ellis J Goodacre R Turner SR (2005) Identification of novel genes in Arabidopsis involved in secondary cell wallformation using expression profiling and reverse genetics Plant Cell 17 2281ndash95
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Caffall KH Pattathil S Phillips SE Hahn MG Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in thebiosynthesis of pectin and xylan in cell walls and seed testa Mol Plant 2 1000ndash14
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Chen L Shi D Zhang W Tang Z Liu J Yang W (2015) The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator inplant cells Nat Commun 6 1ndash10
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Cosgrove DJ (2016) Plant cell wall extensibility Connecting plant cell growth with cell wall structure mechanics and the action of wall-modifying enzymes J Exp Bot 67 463ndash476
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
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Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
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Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
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Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
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Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
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Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
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Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
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Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
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Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
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Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
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Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
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Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
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Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
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Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
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Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
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Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
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Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
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httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
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Dean GH Zheng H Tewari J Huang J Young DS Hwang YT Western TL Carpita NC McCann MC Mansfield SD et al (2007) TheArabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydrationproperties Plant Cell 19 4007ndash4021
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Egelund J Petersen BL Motawia MS Damager I Faik A Olsen CE Ishii T Clausen H Ulvskov P Geshi N (2006) Arabidopsis thalianaRGXT1 and RGXT2 encode Golgi-localized (13)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II Plant Cell 18 2593ndash607
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Foster CE Martin TM Pauly M (2010) Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part IICarbohydrates J Vis Exp 37 e1745
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Fraga D Meulia T Fenster S (2008) Real-Time PCR In SR Gallagher EA Wiley eds Curr Protoc Essent Lab Tech First Edit JohnWiley amp Sons Inc Hoboken pp 1ndash33
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Gille S Haumlnsel U Ziemann M Pauly M (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approachusing hydrolases Proc Natl Acad Sci U S A 106 14699ndash704
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Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Mitros T Dirks W Hellsten U Putnam N et al (2012) Phytozome acomparative platform for green plant genomics Nucleic Acids Res 40 D1178ndashD1186
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Griffiths JSJ Šola K Kushwaha R Lam P Tateno M Young R Voiniciuc C Dean GH Shawn DM Debolt S et al (2015) UnidirectionalMovement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in MucilageExtrusion Adherence and Ray Formation Plant Physiol 168 502ndash520
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Gutierrez L Mauriat M Gunin S Pelloux J Lefebvre J-F Louvet R Rusterucci C Moritz T Guerineau F Bellini C et al (2008) The lackof a systematic validation of reference genes a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants Plant Biotechnol J 6 609ndash618
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Hall BG (2013) Building phylogenetic trees from molecular data with MEGA Mol Biol Evol 30 1229ndash1235Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hao Z Mohnen D (2014) A review of xylan and lignin biosynthesis Foundation for studying Arabidopsis irregular xylem mutants withhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
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Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
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Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
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Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
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Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
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Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
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Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
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Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
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Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
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Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
pleiotropic phenotypes Crit Rev Biochem Mol Biol 9238 1ndash30Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harholt J Suttangkakul A Vibe Scheller H (2010) Biosynthesis of pectin Plant Physiol 153 384ndash95Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Haughn GW Western TL (2012) Arabidopsis Seed Coat Mucilage is a Specialized Cell Wall that Can be Used as a Model for GeneticAnalysis of Plant Cell Wall Structure and Function Front Plant Sci 3 64
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hruz T Laule O Szabo G Wessendorp F Bleuler S Oertle L Widmayer P Gruissem W Zimmermann P (2008) Genevestigator V3 AReference Expression Database for the Meta-Analysis of Transcriptomes Adv Bioinformatics 2008 1ndash5
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Wang X Zhao X Yang X Tang Q He G Zhou G Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 ( IRX14 ) maintainsthe structure of seed coat mucilage in Arabidopsis J Exp Bot 67 1243ndash1257
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hu R Li J Yang X Zhao X Wang X Tang Q He G Zhou G Kong Y (2016b) Irregular xylem 7 (IRX7) is required for anchoring seed coatmucilage in Arabidopsis Plant Mol Biol 92 25ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jacobs A Dahlman O (2001) Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a Nafion-coated probe Anal Chem 73 405ndash410
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Jensen JK Soslashrensen SO Harholt J Geshi N Sakuragi Y Moslashller I Zandleven J Bernal AJ Jensen NB Soslashrensen C et al (2008)Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis Plant Cell 20 1289ndash302
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kester HC Kusters-van Someren M a Muumlller Y Visser J (1996) Primary structure and characterization of an exopolygalacturonasefrom Aspergillus tubingensis Eur J Biochem 240 738ndash746
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kong Y Zhou G Abdeen AA Schafhauser J Richardson B Atmodjo MA Jung J Wicker L Mohnen D Western T et al (2013)GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage Plant Physiol 163 1203ndash17
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li SF Milliken ON Pham H Seyit R Napoli R Preston J Koltunow AM Parish RW (2009) The Arabidopsis MYB5 transcription factorregulates mucilage synthesis seed coat development and trichome morphogenesis Plant Cell 21 72ndash89
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liljebjelke K Adolphson R Baker K Doong RL Mohnen D (1995) Enzymatic synthesis and purification of uridine diphosphate[14C]galacturonic acid a substrate for pectin biosynthesis Anal Biochem 225 296ndash304
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liwanag AJM Ebert B Verhertbruggen Y Rennie EA Rautengarten C Oikawa A Andersen MCF Clausen MH Scheller H V JenniferA et al (2012) Pectin biosynthesis GALS1 in Arabidopsis thaliana is a β-14-galactan β-14-galactosyltransferase Plant Cell 24 5024ndash36
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013Nucleic Acids Res 42 490ndash495
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Kronenberger J Marion-Poll A North HM (2007a) In situ chemical and macromolecular study of the compositionof Arabidopsis thaliana seed coat mucilage Plant Cell Physiol 48 984ndash99
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Macquet A Ralet M-C Loudet O Kronenberger J Mouille G Marion-Poll A North HM (2007b) A naturally occurring mutation in anArabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seedhttpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
mucilage Plant Cell 19 3990ndash4006Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell EG Belshaw NJ Waldron KW Morris VJ (2012) Pectin - An emerging new bioactive food polysaccharide Trends Food SciTechnol 24 64ndash73
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mendu V Griffiths JS Persson S Stork J Downie B Voiniciuc C Haughn GW DeBolt S (2011) Subfunctionalization of cellulosesynthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment Plant Physiol 157 441ndash453
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Moremen KW Ramiah A Stuart M Steel J Meng L Forouhar F Moniz HA Gahlay G Gao Z Chapla D et al (2018) Expression systemfor structural and functional studies of human glycosylation enzymes Nat Chem Biol 14 156ndash162
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Munarin F Tanzi MC Petrini P (2012) Advances in biomedical applications of pectin gels Int J Biol Macromol 51 681ndash689Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mutwil M Obro J Willats WGT Persson S (2008) GeneCAT--novel webtools that combine BLAST and co-expression analyses NucleicAcids Res 36 W320-6
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura A Furuta H Maeda H Takao T Nagamatsu Y (2002) Structural studies by stepwise enzymatic degradation of the mainbackbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan Biosci Biotechnol Biochem 661301ndash13
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Rubtsov D Segura MP Miles GP Stevens TJ Dunkley TPJ Munro S Lilley KS Dupree P (2012) Putativeglycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics Plant Physiol 160 1037ndash51
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nikolovski N Shliaha P V Gatto L Dupree P Lilley KS (2014) Label free protein quantification for plant Golgi protein localisation andabundance Plant Physiol 166 1033ndash43
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Okamura Y Ito S Tadaka S Aoki Y Shirota M Kinoshita K (2014) ATTED-II in 2014 Evaluation of gene coexpression inagriculturally important plants Plant Cell Physiol 55 1ndash7
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Orfila C Soslashrensen SO Harholt J Geshi N Crombie H Truong HN Reid JSG Knox JP Scheller HV (2005) QUASIMODO1 is expressedin vascular tissue of Arabidopsis thaliana inflorescence stems and affects homogalacturonan and xylan biosynthesis Planta 222 613ndash622
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Parsons HT Christiansen K Knierim B Carroll A Ito J Batth TS Smith-Moritz AM Morrison S McInerney P Hadi MZ et al (2012)Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wallbiosynthesis Plant Physiol 159 12ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pattathil S Avci U Baldwin D Swennes AG McGill JA Popper ZA Bootten T Albert A Davis RH Chennareddy C et al (2010) Acomprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies Plant Physiol 153 514ndash25
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pentildea MJ Zhong R Zhou G-K Richardson EA ONeill MA Darvill AG York WS Ye Z-H (2007) Arabidopsis irregular xylem8 and irregularxylem9 implications for the complexity of glucuronoxylan biosynthesis Plant Cell 19 549ndash63
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Caffall KH Freshour G Hilley MT Bauer S Poindexter P Hahn MG Mohnen D Somerville CR (2007) The Arabidopsisirregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan which are essential for secondary cell wall integrityPlant Cell 19 237ndash55
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Persson S Wei H Milne J Page GP Somerville CR (2005) Identification of genes required for cellulose synthesis by regressionanalysis of public microarray data sets Proc Natl Acad Sci U S A 102 8633ndash8638
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pettolino F Walsh C Fincher GB Bacic A (2012) Determining the polysaccharide composition of plant cell walls Nat Protoc 7 1590ndash607Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-C Creacutepeau M-J Vigouroux J Tran J Berger A Salleacute C Granier F Botran L North HM (2016a) The affinity of xylan brancheson rhamnogalacturonan I for cellulose provides the structural driving force for mucilage adhesion to the Arabidopsis seed coat PlantPhysiol pp002112016
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ralet M-CC Crepeau MJ Vigouroux J Tran J Berger A Salle C Granier F Botran L North HM Creacutepeau M-J et al (2016b) XylansProvide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat Plant Physiol 171 165ndash178
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rautengarten C Usadel B Neumetzler L Hartmann J Buumlssis D Altmann T (2008) A subtilisin-like serine protease essential formucilage release from Arabidopsis seed coats Plant J 54 466ndash80
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruprecht C Bartetzko MP Senf D Dallabernadina P Boos I Andersen MCF Kotake T Knox JP Hahn MG Clausen MH et al (2017) ASynthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies Plant Physiol 175 1094ndash1104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B van den Berg W Lokerse A Liao C-Y van Mourik H Moumlller B Peris CL Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants Plant Physiol 156 1292ndash9
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B et al (2012)Fiji an open-source platform for biological-image analysis Nat Methods 9 676ndash682
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desimone M Frommer WB Fluumlgge U-I Kunze R (2003) ARAMEMNONa novel database for Arabidopsis integral membrane proteins Plant Physiol 131 16ndash26
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sessions A Burke E Presting G (2002) High-Throughput Arabidopsis Reverse Genetics System Plant Cell 14 2985ndash2994Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Atmodjo MA Inwood SE Kumar Kolli VS Quigley HF Hahn MG Mohnen D (2006) Functional identification of anArabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase Proc Natl Acad Sci U S A 103 5236ndash5241
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sterling JD Lemons JA Forkner IF Mohnen D (2005) Development of a filter assay for measuring homogalacturonan α-(14)-Galacturonosyltransferase activity Anal Biochem 343 231ndash236
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60 Mol BiolEvol 30 2725ndash2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tan L Eberhard S Pattathil S Warder C Glushka J Yuan C Hao Z Zhu X Avci U Miller JS et al (2013) An Arabidopsis cell wallproteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein Plant Cell 25 270ndash87
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Teh O-K Moore I (2007) An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells Nature 448 493ndash496Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
Uehara Y Tamura S Maki Y Yagyu K Mizoguchi T Tamiaki H Imai T Ishii T Ohashi T Fujiyama K et al (2017) Biochemicalcharacterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall Biochem BiophysRes Commun 486 130ndash136
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Urbanowicz BR Pentildea MJ Moniz HA Moremen KW York WS (2014) Two Arabidopsis proteins synthesize acetylated xylan in vitro PlantJ 80 197ndash206
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Usadel B Kuschinsky A Rosso MG Eckermann N Pauly M (2004) RHM2 is involved in mucilage pectin synthesis and is required forthe development of the seed coat in Arabidopsis Plant Physiol 134 286ndash295
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C (2017) Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Bio-protocol 7 e2323Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Dean GH Griffiths JS Kirchsteiger K Hwang YT Gillett A Dow G Western TL Estelle M Haughn GW (2013) FLYINGSAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seedmucilage Plant Cell 25 944ndash59
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M (2016) Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds Bio-protocol 6 e1801Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Guumlnl M Schmidt MH-W Usadel B (2015a) Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds Plant Physiol 169 2481ndash95
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Pauly M Usadel B (2018) Monitoring Polysaccharide Dynamics in the Plant Cell Wall Plant Physiol 176 2590ndash2600Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Schmidt MH-W Berger A Yang B Ebert B Scheller H V North HM Usadel B Guumlnl M (2015b) MUCILAGE-RELATED10Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage Plant Physiol 169403ndash420
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Yang B Schmidt MH-W Guumlnl M Usadel B (2015c) Starting to Gel How Arabidopsis Seed Coat Epidermal Cells ProduceSpecialized Secondary Cell Walls Int J Mol Sci 16 3452ndash3473
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voiniciuc C Zimmermann E Schmidt MH-W Guumlnl M Fu L North HM Usadel B (2016) Extensive Natural Variation in Arabidopsis SeedMucilage Structure Front Plant Sci 7 803
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Voxeur A Andreacute A Breton C Lerouge P (2012) Identification of putative rhamnogalacturonan-II specific glycosyltransferases inArabidopsis using a combination of bioinformatics approaches PLoS One 7 e51129
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang T Park YB Cosgrove DJ Hong M (2015) Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary CellWalls Evidence from Solid-State Nuclear Magnetic Resonance Plant Physiol 168 871ndash884
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Warde-Farley D Donaldson SL Comes O Zuberi K Badrawi R Chao P Franz M Grouios C Kazi F Lopes CT et al (2010) TheGeneMANIA prediction server biological network integration for gene prioritization and predicting gene function Nucleic Acids Res38 W214-20
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weigel D Glazebrook J (2006) In planta transformation of Arabidopsis CSH Protoc 2006 pdbprot4668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title httpsplantphysiolorgDownloaded on December 2 2020 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-
Western TL Young DS Dean GH Tan WL Samuels L Haughn GW (2004) MUCILAGE-MODIFIED4 Encodes a Putative PectinBiosynthetic Enzyme Developmentally Regulated by APETALA2 TRANSPARENT TESTA GLABRA1 and GLABRA2 in the ArabidopsisSeed Coat Plant Physiol 134 296ndash306
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Winter D Vinegar B Nahal H Ammar R Wilson G V Provart NJ (2007) An electronic fluorescent pictograph Browser for exploringand analyzing large-scale biological data sets PLoS One 2 e718
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ye J Coulouris G Zaretskaya I Cutcutache I Rozen S Madden TL (2012) Primer-BLAST A tool to design target-specific primers forpolymerase chain reaction BMC Bioinformatics 13 134
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Young RE McFarlane HE Hahn MG Western TL Haughn GW Samuels L (2008) Analysis of the Golgi apparatus in Arabidopsis seedcoat cells during polarized secretion of pectin-rich mucilage Plant Cell 20 1623ndash38
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yu L Shi D Li J Kong Y Yu Y Chai G Hu R Wang J Hahn MG Zhou G (2014) CSLA2 a Glucomannan Synthase is Involved inMaintaining Adherent Mucilage Structure in Arabidopsis Seed Plant Physiol 164 1842ndash1856
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on December 2 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Parsed Citations
-