1 short title: scyl2 genes play a role in plant cell growth · 2017/7/27 · 149 and 21 to 27%...
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Short title: SCYL2 genes play a role in plant cell growth 1
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Corresponding author(s) information: 3
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Daniel P. Schachtman, Ph.D. 5
Professor and Director of Center for Biotechnology 6
University of Nebraska Lincoln 7
Department of Agronomy and Horticulture 8
Member of Center for Plant Science Innovation 9
Beadle Center E243, Lincoln, NE 68588 10
1-402-472-1448 (office) 11
1-314-799-2427 (cell) 12
E-mail address: [email protected] 13
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Ji-Yul Jung, Ph.D. 15
Postdoctoral Researcher 16
Department of Plant Molecular Biology (DBMV) 17
University of Lausanne 18
UNIL–Sorge, Biophore Building 19
1015 Lausanne, Switzerland 20
41-21-692-4239 (office) 21
41-78-898-6322 (cell) 22
E-mail address: [email protected] 23
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Plant Physiology Preview. Published on July 27, 2017, as DOI:10.1104/pp.17.00824
Copyright 2017 by the American Society of Plant Biologists
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Article Title: 25
SCYL2 genes are involved in clathrin-mediated vesicle trafficking 26
and essential for plant growth 27
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Ji-Yul Jung*, Dong Wook Lee, Stephen Beungtae Ryu, Inhwan Hwang, and Daniel P. 29
Schachtman* 30
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Department of Plant Molecular Biology, University of Lausanne, Switzerland (J.J); Division 32
of Integrative Biosciences and Biotechnology, Pohang University of Science and 33
Technology, Pohang 790-784, Korea (D.W.L., I.H.); Plant Systems Engineering Research 34
Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, 35
Korea (S.B.R.); Department of Agronomy and Horticulture, University of Nebraska Lincoln, 36
Lincoln, NE 68588, USA (D.P.S.) 37
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One sentence summary: 39
Vesicle membrane-associated SCYL2 proteins are vital for plant development. 40
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Footnotes 42
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Author contributions: 44
J.J., D.P.S., D.W.L., S.B.R., and I.H. conceived and designed the experiments. J.J. and 45
D.W.L. performed the experiments. J.J. and D.W.L. analysed the data., J.J., D.P.S., .S.B.R, 46
and I.H. wrote the manuscript. 47
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Funding information: 49
D.W.L and I.H. were supported by the Cooperative Research program for Agriculture 50
Science & Technology Development (Project No. 010953012016), Rural Development 51
Administration, Republic of Korea, S.B.R. was supported by grant (PJ01109103) from the 52
Next-Generation BioGreen 21 Program (SSAC), Korea Rural Development Administration. 53
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Corresponding author emails: 55
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*Address correspondence to: Ji-Yul Jung ([email protected]) and Daniel P. 56
Schachtman ([email protected]) 57
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ABSTRACT 60
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Protein transport between organelles is an essential process in all eukaryotic cells and is 62
mediated by the regulation of processes such as vesicle formation, transport, docking and 63
fusion. In animals, SCY1-Like2 (SCYL2) binds to clathrin and has been shown to play 64
roles in trans-Golgi network (TGN)-mediated clathrin coated vesicle (CCV) trafficking. Here 65
we demonstrate that SCYL2A and SCYL2B, which are Arabidopsis homologs of animal 66
SCYL2, are vital for plant cell growth and root hair development. Studies of the SCYL2 67
isoforms using multiple single or double loss of function alleles show that SCYL2B is 68
involved in root hair development and that SCYL2A and SCYL2B are essential for plant 69
growth and development and act redundantly in those processes. The quantitative RT-70
PCR and β-Glucuronidase (GUS)-aided promoter assay show that SCYL2A and SCYL2B 71
are differentially expressed in various tissues. We also show that SCYL2 proteins localize 72
to the Golgi, TGN, and pre-vacuolar compartment (PVC) and co-localize with clathrin 73
heavy chain1 (CHC1). Furthermore, bimolecular fluorescence complementation (BiFC) 74
and co-immunoprecipitation data show that SCYL2B interacts with CHC1 and two Soluble 75
NSF Attachment protein Receptors (SNAREs): Vesicle Transport through t-SNARE 76
Interaction11 (VTI11) and VTI12. Finally, we present evidence that the root hair tip 77
localization of Cellulose Synthase-Like D3 (CSLD3) is dependent on SCYL2B. These 78
findings suggest the role of SCYL2 genes in plant cell developmental processes via 79
clathrin-mediated vesicle membrane trafficking. 80
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Keywords : Arabidopsis; clathrin-mediated vesicle trafficking; plant development; SCYL2, 82
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INTRODUCTION 84
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Vesicle-mediated protein transport between organelles is a fundamental process in all 86
eukaryotic cells. In many cases, newly synthesized proteins in the endoplasmic reticulum 87
(ER) are targeted to specific membranes where they function with optimal activity (Jurgens, 88
2004; Hwang and Robinson, 2009; Robinson and Neuhaus, 2016). Protein targeting 89
involves processes including the formation, transport, docking and fusion of vesicles. A 90
number of proteins such as coat proteins, ADP-ribosylation factor, and SNAREs are also 91
required for the regulation of the vesicle trafficking (Jurgens, 2004; Bassham and Blatt, 92
2008; Nielsen et al., 2008; Chen et al., 2011; Fan et al., 2015; Paez Valencia et al., 2016). 93
Depending on the species of coat proteins, vesicles can be classified into three 94
major types: coat protein-1 (COPI), coat protein-2 (COPII), and CCV. Among them, CCV is 95
formed by clathrin triskelia consisting of three clathrin heavy chains (CHCs) and three 96
clathrin light chains (CLCs). The Arabidopsis genome contains two CHC genes (CHC1 97
and CHC2) and three CLC genes (CLC1, CLC2, and CLC3) (Kitakura et al., 2011; Wang 98
et al., 2013; Paez Valencia et al., 2016). In plants CCV mediates protein trafficking from 99
the TGN to the multivesicular PVC, which fuses with the vacuole for the degradation of 100
proteins (Song et al., 2006; Hwang and Robinson, 2009; Park et al., 2013; Paez Valencia 101
et al., 2016; Robinson and Neuhaus, 2016). CCV is also known to be involved in the 102
endocytosis of proteins such as PIN-FORMED (PIN) auxin efflux carriers and 103
Brassinosteroid Insensitive1 (BRI1) receptor kinase at the plasma membrane (PM) 104
(Kitakura et al., 2011; Di Rubbo et al., 2013; Wang et al., 2013). Recently, a plant specific 105
TPLATE adaptor protein complex is shown to be essential for clathrin-mediated 106
endocytosis (Gadeyne et al., 2014; Wang et al., 2016). However, the CCV machinery in 107
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plants is still poorly known when compared to the CCV components identified in animals 108
(Chen et al., 2011; Paez Valencia et al., 2016). 109
In animals, SCY1-LIKE 2 (SCYL2), which belongs to the SCY1-like gene family, 110
was identified as a component of the CCVs (Conner and Schmid, 2005). It was shown that 111
animal SCYL2 directly binds to both clathrin and Adaptor Protein-2 (AP2) which is an 112
adaptor protein (Conner and Schmid, 2005; Duwel and Ungewickell, 2006). SCYL2 has 113
been suggested to function as a kinase (Conner and Schmid 2005) and is found at the 114
Golgi, TGN, and endosomes in animal systems (Duwel and Ungewickell, 2006; Borner et 115
al., 2007). Moreover, SCYL2 selectively induces lysosomal degradation of Frizzled5 116
(Fzd5) which is a PM receptor involved in the Wnt signaling pathway (Terabayashi et al., 117
2009). It has also been reported that lysosomal hydrolase cathepsin D is mis-sorted in 118
SCYL2-depleted cells (Duwel and Ungewickell, 2006) and that knockdown of SCYL2 in 119
Xenopus tropicalis causes severe developmental defects (Borner et al., 2007). Recently, it 120
was shown that SCYL2 is a key regulator of neuronal function and survival in mouse brain 121
(Gingras et al., 2015; Pelletier, 2016). These data indicate that in animals SCYL2 functions 122
in clathrin-mediated vesicle trafficking between the TGN and the endosomal system with 123
an important role in cell developmental processes. 124
In this study, two Arabidopsis homologs, SCYL2A and SCYL2B, are shown to play 125
important roles in plant cell growth. SCYL2B is involved in root hair developmental 126
processes and both SCYL2A and SCYL2B are essential for plant growth and act 127
redundantly in certain developmental processes. GUS-aided promoter assay indicated that 128
SCYL2A and SCYL2B are differentially expressed in various tissues. Moreover, we found 129
that SCYL2B localizes at the Golgi, TGN, and PVC and interacts with CHC1 and two 130
SNAREs: VTI11 and VTI12. Finally, we show that SCYL2B co-localizes with SCYL2A and 131
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CHC1 and is required for the tip localization of CSLD3 in root hairs. These data suggest 132
that the plant SCYL2 genes, new components of CCV trafficking in plants, play vital roles 133
in plant cell developmental processes. 134
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RESULTS 137
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SCYL2 is Conserved in Many Eukaryotes 139
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In Arabidopsis, there are two SCYL2 genes: SCYL2A and SCYL2B which encode 913 and 141
909 amino acids, respectively. These two genes likely arose from a genome duplication, 142
as the two genes fall into two syntenic chromosomal regions (Supplemental Fig. S1; Plant 143
Genome Duplication Database, http://chibba.agtec.uga.edu/duplication/index/home; (Tang 144
et al., 2008)). Sequence alignment and phylogenetic analysis show that SCYL2 genes are 145
conserved in the genomes of many eukaryotes (Fig. 1, A and B). Comparison of amino 146
acid sequences of full-length Arabidopsis SCYL2 proteins revealed 57 to 83 % amino acid 147
identity to proteins in other plant species such as papaya, poplar, soybean, corn, and rice, 148
and 21 to 27% amino acid identity to the SCYL proteins in animal species such as human, 149
chimpanzee, dog, cow, mouse, rat, chicken, fruit fly, and mosquito (Fig. 1A). 150
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SCYL2B Functions in Root Hair Development 152
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To characterize the functions of SCYL2 genes in Arabidopsis, we identified several T-DNA 154
insertional mutant lines of SCYL2A and SCYL2B from Salk and Sail lines. Three scyl2a 155
alleles and four scyl2b alleles were obtained (Fig. 2). RT-PCR analysis confirmed the 156
absence of full-length transcripts of SCYL2A or SCYL2B in scyl2a or scyl2b alleles except 157
for scyl2b-2, which has a decreased amount of full-length transcripts due to the T-DNA 158
insertion of second intron in SCYL2B (Fig. 2, B and C). Four scyl2a scyl2b double mutants 159
were obtained from the crosses between scyl2a and scyl2b alleles (Fig. 2B) and RT-PCR 160
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analysis showed that full-length transcripts of both SCYL2A and SCYL2B were absent in 161
all scyl2a scyl2b double mutants (Fig. 2C). 162
To investigate the possible role of SCYL2A and SCYL2B in plants, we analyzed 163
the phenotype of scyl2a, scyl2b, and scyl2a scyl2b double mutant alleles. Three days after 164
germination, the root hairs of scyl2b alleles and scyl2a scyl2b double mutants were shorter 165
than wild type (P < 0.05; Fig. 3, A and B left) and were as short as rhd2-4, which has been 166
shown to be a root hair defective mutant (P < 0.05; Fig. 3, A and B right). The scyl2b 167
alleles also had abnormal root hair phenotypes such as v shape, y shape, wavy shape, 168
and bulged shape (Fig. 3C). The percentage of aberrant root hairs per root was much 169
higher in scyl2b mutants than the wild type (P < 0.05; Fig. 3C). There were no defects of 170
root hairs in scyl2a alleles (Fig. 3, A and B). These data indicate that SCYL2B plays an 171
important role in polarized root hair growth. 172
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SCYL2A and SCYL2B are Essential for Plant Growth 174
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We further analyzed the phenotypes of roots and shoots in scyl2a, scyl2b, and scyl2a 176
scyl2b alleles. Ten days after germination scyl2a scyl2b double mutants had significantly 177
shorter primary roots and smaller shoots (P < 0.05; Fig. 4, A, B, and C) while scyl2a or 178
scyl2b single mutants had no difference in roots and shoots compared to wild type (Fig. 4, 179
A, B, and C). One month-old scyl2a scyl2b double mutants grown in soil showed a striking 180
tiny shoot phenotype (Fig. 4D). These data indicate that both SCYL2A and SCYL2B are 181
involved in plant growth and development and redundant in certain aspects of their 182
function. 183
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SCYL2A and SCYL2B Expression Levels in Various Tissues 185
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To determine in which developmental stages or tissues in Arabidopsis SCYL2A and 187
SCYL2B are highly expressed, we analyzed gene expression levels in various tissues of 188
seven day-old seedlings and one month-old plants. Quantitative RT-PCR analysis showed 189
that both SCYL2A and SCYL2B are co-expressed in all tissues tested such as roots, 190
rosette leaves, cauline leaves, stems, unopened flowers, opened flowers, and siliques (Fig. 191
5A) and that the expression of SCYL2B was about two- to three-fold higher compared to 192
that of SCYL2A (Fig. 5A). The expression level of both SCYL2A and SCYL2B was very 193
low in siliques (Fig. 5A). 194
To analyze the tissue specific expression pattern of SCYL2A and SCYL2B in 195
Arabidopsis, we monitored the GUS expression driven by the promoter of SCYL2A and 196
SCYL2B in transgenic plants. In plants carrying SCYL2Apromoter:GUS, GUS staining was 197
mainly detected in leaf mesophyll cells while very week GUS staining was seen in root 198
vascular tissues (Fig. 5B). In plants carrying SCYL2Bpromoter:GUS, strong GUS staining 199
was detected in both shoot and root vascular tissues, in root cap regions including 200
columella cells and lateral root caps, and also in some tissues of specialized cell types 201
such as trichomes, guard cells, and root hairs (Fig. 5B). 202
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SCYL2B is Localized at The Golgi, TGN, and PVC. 204
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To define the subcellular localization of SCYL2B in Arabidopsis, transgenic plants carrying 206
35S:YFP:SCYL2B were analyzed. YFP:SCYL2B was localized to the tips of growing root 207
hair cells (Fig. 6A and Supplemental Fig. S2). However, in mature root hairs where tip 208
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growth was finished, the tip-localization pattern of YFP:SCYL2B disappeared (Fig. 6B and 209
Supplemental Fig. S2). In addition, YFP:SCYL2B appeared to be localized at some 210
aggregated endosomal structures (Fig. 6B). In animals, a clathrin-coated vesicle-211
associated kinase of 104 kDa (CVAK104), an Arabidopsis SCYL2A/B otholog, plays an 212
important role in vesicle trafficking and is localized at the Golgi, TGN and endosomes 213
(Duwel and Ungewickell, 2006; Borner et al., 2007). To determine subcellular localization 214
of SCYL2B, colocalization studies were performed using several fluorescent protein 215
markers for subcellular organelles such as the Golgi, TGN, PVC, PM, mitochondria, 216
plastids, and peroxisomes. In Arabidopsis leaf protoplasts and leaf epidermal cells, 217
YFP:SCYL2B was co-localized with markers for the Golgi, TGN, and PVC but not with 218
markers for mitochondria, plastids, peroxisomes, and PM (Fig. 6, Supplemental Fig. S3 219
and S4). The percentages of YFP:SCYL2B-positive spots overlapping with the Golgi, TGN, 220
and PVC organelle marker proteins were about 38%, 37%, and 26%, respectively 221
(Supplemental Table S1). The localization data of SCYL2B suggest that SCYL2B is 222
membrane localized and may be involved in vesicle membrane trafficking in plant cells. 223
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SCYL2B Interacts with CHC1, VTI11, and VTI12. 225
226
In animals, SCYL2 acts as a component of CCVs and can interact with clathrin (Conner 227
and Schmid, 2005; Duwel and Ungewickell, 2006). To determine whether or not 228
Arabidopsis SCYL2 proteins can also bind to components of CCVs, we conducted a split 229
YFP-based bimolecular fluorescence complementation assay (BiFC). To make constructs 230
for BiFC, the N-terminal fragment of YFP (nYFP) was fused to the N-terminus of SCYL2B 231
and the C-terminal fragment of YFP (cYFP) was fused to the N-terminus of putative 232
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SCYL2B binding candidates such as CHC1, VTI11, and VTI12. The nYFP:SCYL2B 233
construct was transiently co-expressed with cYFP fusion constructs mentioned above in 234
Nicotiana benthamiana leaf epidermal cells via agrobacterium infiltration. Clear YFP 235
fluorescence signal showing a punctate staining pattern was detected when 236
nYFP:SCYL2B was transiently co-transformed with cYFP:CHC1, cYFP:VTI11, or 237
cYFP:VTI12 (Fig. 7A). There was no YFP fluorescence signal when nYFP:SCYL2B was 238
transiently co-transformed with cYFP (Fig. 7A). Western blotting analysis using polyclonal 239
anti-GFP antibody showed that cYFP:CHC1, cYFP:VTI11, and cYFP:VTI12 were co-240
expressed well with nYFP:SCYL2B in the tobacco leaf cells (Supplemental Fig. S5). As 241
well as BiFC experiments, we also conducted co-immunoprecipitation experiments to 242
confirm the protein interaction of BiFC results. HA:CHC1, HA:VTI11, and HA:VTI12 were 243
transiently co-expressed with YFP and YFP:SCYL2B in tobacco leaf cells and then the 244
HA-tagged proteins were pulled down by anti-HA beads and probed by anti-GFP antibody. 245
YFP:SCYL2B was detected in the bound fraction of HA:CHC1, HA:VTI11, and HA:VTI12 246
whereas YFP alone was not (Fig. 7B and Supplemental Fig. S7) indicating that SCYL2B 247
interacts with CHC1, VTI11, and VTI12. We also tested the protein interaction by pulling 248
down YFP-tagged proteins first, followed by the detection of HA tagged proteins since 249
commercial anti-GFP beads called GFP-trap were also available. When YFP and 250
YFP:SCYL2B were immunoprecipitated by GFP-trap beads and then probed by anti-HA 251
antibody, HA:CHC1, HA:VTI11, and HA:VTI12 were detected in the bound fraction of 252
YFP:SCYL2B (Supplemental Fig. S6 and S7) confirming the protein interaction. However, 253
a weak signal of HA:VTI11 and HA:VTI12 was also detected in the bound fraction of YFP 254
alone despite thorough washing of beads indicating that VTI11 and VTI12 tend to bind 255
GFP-trap beads non-specifically (Supplemental Fig. S6 and S7). Taken together, these 256
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data indicate that Arabidopsis SCYL2B interacts with a clathrin and two v-SNAREs: VTI11 257
and VTI12 in vivo. 258
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SCYL2B Co-localizes with SCYL2A and CHC1 and is Required for Tip Localization of 260
CSLD3 in Root Hairs. 261
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We determined the localization of SCYL2 proteins by tagging at the N- or C-terminus with 263
fluorescent proteins GFP and RFP. When N-terminal GFP tagged SCYL2B and C-terminal 264
RFP tagged SCYL2B were transiently co-expressed in tobacco leaf epidermal cells, there 265
was no difference in the localization of these proteins (Fig. 8A) suggesting that tagging 266
fluorescent proteins to SCYL2 proteins may not alter their subcellular localization. We also 267
compared the localization of GFP:SCYL2B with SCYL2A:RFP by coexpressing them in 268
tobacco cells and the results showed that there was no difference in their subcellular 269
localization (Fig. 8B). The co-localization of SCYL2A with SCYL2B suggests that SCYL2A 270
may act in a similar fashion as SCYL2B probably locating at Golgi, TGN, and PVC. 271
Since BiFC and co-immunoprecipitation data showed the interaction between 272
SCYL2B and CHC1, we also examined if they co-localize. We found that both SCYL2A 273
and SCYL2B co-localized well with CHC1 (Fig. 8, C and D) when they were co-expressed 274
transiently in tobacco leaf epidermal cells, supporting the notion that SCYL2 proteins are 275
involved in clathrin-mediated vesicle trafficking. 276
It was reported that the function of root hair tip-localized proteins such as RHO-277
RELATED PROTEIN FROM PLANTS 2 (ROP2), ROOT HAIR DEFECTIVE 2 (RHD2), 278
RAB GTPASE HOMOLOG A 4B (RABA4B), SYNTAXIN OF PLANTS 123 (SYP123), 279
PROLINE-RICH PROTEIN 3 (PRP3), and CSLD3 is important for the proper root hair tip 280
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growth (Jones et al., 2002; Foreman et al., 2003; Preuss et al., 2004; Park et al., 2011; 281
Ichikawa et al., 2014; Rodriguez-Furlan et al., 2016). To understand the root hair tip 282
growth pathways in which SCYL2B functions, Col-0 and scyl2b mutants were transformed 283
with GFP fusion constructs including ROP2, RHD2, RABA4B, SYP123, PRP3, and CSLD3 284
genes whose expression is driven by EXPANSIN A7 (EXPA7) promoter, a root hair 285
specific promoter (Kim et al., 2006). Interestingly, the analysis of transgenic lines showed 286
that only CSLD3 is mislocalized in the root hairs of scyl2b mutants (Fig. 9 and 287
Supplemental Fig. S8) indicating that SCYL2B is important for mediating the tip 288
localization of CSLD3 proteins in root hairs. 289
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DISCUSSION 292
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SCYL2 is a member of the SCY1-Like protein family. SCY1 is a kinase-like protein 294
identified in Saccharomyces cerevisiae. In animals, there are three SCY1-LIKE genes: 295
SCYL1, SCYL2, and SCYL3. SCYL1 is the gene responsible for the neurodegenerative 296
mouse model mdf and plays important roles in COPI-mediated retrograde trafficking from 297
the Golgi to the ER and the regulation of the Golgi morphology (Burman et al., 2010). 298
SCYL2 is involved in the TGN-mediated CCV membrane trafficking. SCYL3 binds ezrin 299
which is a protein that mediates the interaction between transmembrane proteins and actin 300
and is implicated in regulating cell adhesion/migration complexes in migrating cells 301
(Sullivan et al., 2003). In plants there are othologs only for the animal SCYL1 and SCYL2. 302
In Arabidopsis, the gene which encodes AT2G40730 corresponds to the ortholog of animal 303
SCYL1 and two loci (AT1G22870 and AT1G71410) described in this study correspond to 304
the orthologs of animal SCYL2. 305
We showed that Arabidopsis SCYL2B localizes at the Golgi, TGN, and PVC and 306
co-localizes with SCYL2A and CHC1 (Fig. 6 and 8). Moreover, BiFC and co-307
immunoprecipitation assay showed that SCYL2B binds to CHC1 and two v-SNAREs: 308
VTI11 and VTI12 (Fig. 7). Interestingly, the analysis of SCYL2 protein sequence alignment 309
revealed a conserved clathrin binding motif (SLLDLL) at the very end of C-terminus on 310
both SCYL2A and SCYL2B (Lafer, 2002). However, based on BiFC analysis, it turned out 311
that this binding motif is not essential for the interaction between SCYL2B and CHC1 312
(Supplemental Fig. S9). For the co-immunoprecipitation experiments, we used YFP alone 313
as a negative control and YFP alone did not bind CHC, VTI11, and VTI12. Moreover, there 314
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was no precipitation of SCYL2B in the control samples where HA-tagged proteins are 315
absent (Supplemental Fig. S7). We also had BiFC data showing that certain membrane 316
proteins such as adaptor proteins, which are involved in vesicle membrane trafficking, did 317
not interact with SCYL2B (Supplemental Fig. S10). These findings suggest that the 318
interaction between SCYL2B and proteins tested in this study is specific. 319
Our data showing SCYL2 localization and interaction indicate that Arabidopsis 320
SCYL2 proteins play a role in CCV-mediated membrane trafficking and also suggest that 321
plant SCYL2 may act in the similar manner as animal SCYL2. Animal SCYL2 is well 322
known to interact with clathrin. However, there is no report showing the interaction of 323
animal SCYL2 with SNAREs. Thus, our data showing the interaction of Arabidopsis 324
SCYL2B with two v-SNAREs are a novel finding. In animals, SCYL2 has been also 325
suggested to function as a SNARE adaptor (Hirst et al., 2015) due to the fact that there is 326
a long adaptor-like structure with a predicted coiled-coil domain at the C-terminus of 327
SCYL2 (Pelletier, 2016) and that knockdowns of animal SCYL2/CVAK104 result in 328
reduced levels of syntaxin 8 and vti1b associated with CCVs (Borner et al., 2007). These 329
findings along with our new data showing the interaction of AtSCYL2B with two v-SNAREs 330
suggest that plant SCYL2 may act as a SNARE adaptor in clathrin-mediated vesicle 331
trafficking. 332
In BiFC assays, a punctate staining pattern of YFP fluorescence was seen when 333
SCYL2B was transiently co-expressed with CHC1, VTI11, and VTI12 (Fig. 7A). Moreover, 334
a similar fluorescence pattern was observed in the co-localization studies with SCYL2 335
proteins and CHC1 (Fig. 8). Previously, it was shown that clathrin localizes at TGN and 336
PM (Blackbourn and Jackson, 1996; Dhonukshe et al., 2007; Fujimoto et al., 2010; Van 337
Damme et al., 2011) and that VTI11 localizes primarily to the TGN, with a minor portion 338
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localized at PVC (Zheng et al., 1999; Bassham et al., 2000; Surpin et al., 2003). VTI12 is 339
also used as a TGN marker (Geldner et al., 2009). Likewise, the treatment of brefeldin A 340
(BFA) and wortmannin, endomembrane trafficking inhibitors (Hicks and Raikhel, 2010; 341
Mishev et al., 2013), induced the formation of BFA compartments and ring-like structures 342
of SCYL2B proteins (Supplemental Fig. S11). Based on the co-localization data and 343
chemical inhibitor assay, it is likely that SCYL2 proteins function with clathrin, VTI11, and 344
VTI12 mainly at TGN and/or PVC. 345
Since the localization of SCYL2 proteins was obtained by transient overexpression 346
in Arabidopsis and tobacco leaf epidermal cells, we cannot completely rule out the 347
possibility that the localization of endogenous SCYL2 could be different to that of 348
overexpressed proteins. However, we believe that the localization of SCYL2 based on 349
transient overexpression represents the actual localization of SCYL2 in the cell for the 350
following reasons. First, when SCYL2 was expressed in tobacco cells using the native 351
SCYL2B promoter or EXPA7 promoter, the localization pattern of SCYL2 was similar to 352
that of overexpressed SCYL2 (Supplemental Fig. S12). Second, the fact that animal 353
SCYL2 localizes to the Golgi, TGN and endosomes is also consistent with our findings 354
about SCYL2 localization. Lastly, the protein interaction and colocalization data showing 355
the functional relationship of SCYL2 proteins with CHC1, VTI11 and VTI12, which mainly 356
localize to the TGN and/or the PVC, also support our localization data. 357
We showed that the loss of function of SCYL2B leads to the formation of both 358
short root hairs and morphologically abnormal root hairs such as wavy hairs and branched 359
hairs (Fig. 3) indicating that SCYL2 is involved in both root hair elongation and polar tip 360
growth of root hairs. SCYL2B was located to the tips of growing root hair cells and the tip-361
localization pattern of SCYL2B disappeared in mature root hairs (Fig. 6A, 6B, and 362
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18
Supplemental Fig. S2) providing further evidence for the role of SCYL2B in polar tip growth 363
of root hairs. Active membrane trafficking processes govern the polar tip growth of root 364
hairs by the regulation of endocytosis and exocytosis (Ishida et al., 2008; Lee and Yang, 365
2008). Based on the observation that SCYL2B localizes at the Golgi, TGN, and PVC (Fig. 366
6) and the fact that SCYL2B binds to and co-localizes with clathrin and SNAREs (Fig. 7 367
and 8), it is possible that SCYL2B is involved in CCV-mediated membrane trafficking in 368
polar tip growth of root hairs. In plants, the Golgi and TGN/EE play a critical role in the 369
trafficking of secretory, endocytic, and vacuolar vesicles and are also important for root 370
hair development (Hwang and Robinson, 2009). For example, RHD2, a NADPH oxidase 371
which produces ROS, is known to play an important role in root hair elongation. It was 372
shown that vesicle trafficking mediated by the Golgi and TGN/early endosomes (EE) is 373
essential for both PM targeting and function of RHD2 indicating that the proper function of 374
the Golgi and TGN/EE is important for root hair elongation (Takeda et al., 2008; Lam et al., 375
2009). The function of PVC is also known to be important for root hair tip growth. It was 376
shown that LY294002, a phosphatidylinositol 3-kinase (PI3K)-specific inhibitor, inhibits the 377
fusion of PVC/late endosomes with vacuoles leading to the significant reduction of root 378
hair length (Lee et al., 2008) supporting the role for PVC in root hair elongation. Thus, the 379
localization of SCYL2B on the Golgi, TGN, and PVC and its root hair phenotype also 380
support the roles of these endomembrane systems in the development of root hairs. 381
The localization of proteins essential for root hair tip growth indicated that SCYL2B 382
is not involved in the vesicle trafficking pathways for RHD2 and SYP123, which are 383
membrane proteins localized at the root hair tip. However, the mis-localization of CSLD3, a 384
membrane protein, in root hairs of scyl2b mutants showed that SCYL2B plays a role in the 385
vesicle trafficking pathway that mediates the PM localization of CSLD3 at the root hair tip. 386
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19
Previously, it was reported that CSLD3 is localized at the Golgi, ER, and PM (Favery et al., 387
2001; Bernal et al., 2008; Zeng and Keegstra, 2008; Park et al., 2011) and it was 388
suggested that cellulose synthases (CesAs), CSLD3 homologs, may be trafficked to the 389
plasma membrane via the Golgi and TGN (Schneider et al., 2016). Taken together, these 390
findings suggest that SCYL2B may be involved in the Golgi/TGN-mediated 391
exocytosis/secretion of vesicles containing CSLD3. To determine whether SCYL2B 392
proteins are involved in endocytosis processes, we also performed uptake assay using an 393
endocytic tracer N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) 394
Hexatrienyl) Pyridinium Dibromide (FM4-64) in root hair cells of wild type and scyl2a 395
scyl2b double mutants and found no difference in FM4-64 uptake (Supplemental Fig. S13). 396
It was reported that FM4-64 uptake decreased only in chc2 mutants but not in chc1 397
mutants (Kitakura et al., 2011). Thus, it is unlikely that SCYL2 proteins, whose function 398
may be associated with CHC1 based on protein interaction studies, are involved in 399
endocytosis. Overall, based on both the subcellular localization and the binding 400
characteristics of SCYL2B proteins, we suggest that SCYL2B may act as a component of 401
clathrin-mediated vesicle membrane trafficking that regulates exocytosis/secretion 402
mediated by TGN/EE and PVC in the process of root hair tip growth. 403
We also showed that plant shoot and root growth is severely reduced in scyl2a 404
scyl2b double mutants while the only observable phenotype in the single mutants was a 405
root hair defect in scyl2b (Fig. 3 and 4). This indicates that SCYL2A and SCYL2B act 406
redundantly in most tissues and play a critical role in processes other than root hair 407
development such as plant cell growth. However, although the phenotypic data using 408
mutants indicate that there is some functional redundancy between SCYL2A and SCYL2B, 409
the GUS expression pattern suggests that the function of SCYL2A and SCYL2B is 410
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20
somewhat specified to certain tissues. For example, based on GUS staining data, it seems 411
that SCYL2A has a dominant role in leaf mesophyll cells whereas SCYL2B has a dominant 412
role in vascular tissues and in certain specialized cell types such as trichomes, guard cells, 413
and root hairs where lipid membrane change occurs actively. Since GUS expression data 414
showed that SCYL2 proteins are expressed in root columella cells, trichomes, and guard 415
cells, we examined scyl2 mutants more carefully for the phenotype of these tissues and 416
found abnormal morphology of root columella cells only in scyl2a scyl2b double mutants 417
suggesting a role of SCYL2 proteins in the development of root columella cells 418
(Supplemental Fig. S14). No phenotypic difference is found in the cell types of guard cells 419
and trichomes (Data not shown). We also examined the shoot and root phenotypes of 420
several SCYL2B, HA-SCYL2B and YFP-SCYL2B overexpression lines and there were no 421
phenotypic differences in these lines when compared to WT (data not shown). 422
In animals, SCYL2 is also known as a clathrin-coated vesicle-associated kinase of 423
104 kDa (CVAK104) and has a putative Ser/Thr protein kinase domain at its N-terminus 424
(Conner and Schmid, 2005). However, animal and plant SCYL2 genes lack a number of 425
highly conserved catalytic residues within the putative kinase domain such as aspartic acid 426
which is essential in other kinases (Manning et al., 2002). It is still controversial whether or 427
not the SCYL2 has kinase activity. It was reported that in the presence of poly-L-lysine, 428
SCYL2 was able to autophosphorylate and to phosphorylate the β2-adaptin subunit of AP2 429
(Conner and Schmid, 2005) indicating that SCYL2 has an ability to phosphorylate some 430
proteins under certain circumstances. However, in another report, kinase activity was not 431
detected in immunoprecipitated or bacterially expressed SCYL2/CVAK104 (Duwel and 432
Ungewickell, 2006). We tried to express the several recombinant forms of Arabidopsis 433
SCYL2B with 6xHis- or GST- tags in E.coli to find out whether it had a kinase activity, but 434
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21
failed to express the protein possibly due to the instability of the protein. In a systematic 435
comparison study using 1,019 Arabidopsis protein kinases, Arabidopsis SCYL2 genes 436
have been classified as unclustered kinases (Wang et al., 2003). It may be possible to 437
speculate that Arabidopsis SCYL2 proteins may be kinases that regulate membrane 438
trafficking by phosphorylating CCV components. More detailed studies such as phospho-439
proteomic approaches using mutant alleles and mutagenesis approaches of the putative 440
kinase domain will be required to demonstrate that SCYL2 is a protein kinase. 441
In conclusion, the data presented here provide evidence that SCYL2 genes play 442
an important role in plant cell growth and may be involved in clathrin-mediated vesicle 443
trafficking. Additional studies will be required to pinpoint the precise function of the SCYL2 444
genes. The identification of other binding partners of the SCYL2 proteins and investigation 445
about the possibility that SCYL2 proteins act as a kinase will be a next step towards 446
revealing the detailed roles of the SCYL2 genes in plant membrane trafficking and 447
developmental processes. 448
449
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22
MATERIALS AND METHODS 450
451
Plant Materials and Growth Conditions 452
453
Arabidopsis plants used in this study were in the Columbia background. Seeds were 454
sterilized in 70% (v/v) ethanol and 0.05% (v/v) Triton X-100 and then planted on 10 cm-455
diameter sterile plates containing ammonium free complete nutrient agar medium (Jung et 456
al., 2009). After stratification of the seeds at 4°C for 2~3 days, the plates were transferred 457
to the growth chamber at 22°C with a 16-h daylength at 200 µmol·m–2s–1. Seedlings were 458
grown vertically. Three- to 10-day-old seedlings grown on plates were used throughout the 459
study. In addition, plants were also grown on soil under the same conditions. Unless 460
otherwise stated, all experiments in this study were repeated at least twice with similar 461
results. 462
463
Characterization of T-DNA Insertion Mutants and RT-PCR 464
465
T-DNA Insertional Salk and Sail lines were obtained from the ABRC stock center 466
(Columbus, Ohio). Homozygous Salk and Sail lines for scyl2a and scyl2b alleles were 467
isolated by PCR-based genotyping using the following primers: 5’-468
CAAACCACATGAAGCTCAGTATTC-3’ and 5’-GATTATCTAAAGCTGCTGAAGATGC-3’ for 469
scyl2a-1, 5’-CTCGATGGTCAGGTAATGCTTGATAC-3’ and 5’-470
GTTGGTACTGATGTCCGGTTCGATGACT-3’ for scyl2a-2, 5’-471
CAGGAAAATAGAGGAAAAAAGAGGAGTT-3’ and 5’-472
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23
TCACAATAGATCTAACAAAGATGGCTGT-3’ for scyl2a-3, 5’- 473
GCATGTTATCCAAATTCCCAGCCTG-3’ and 5’-474
AACAAGTTGCAGAAAGAGAGAGAGGTG-3’ for scyl2b-1, 5’-475
GCGCTTGGTAATGTTGAGAATGTGG-3’ and 5’-AGTGGAACGATTGCATGTTATCCA-3’ 476
for scyl2b-2, 5’-TCCGAAGTGACGCTAGGTTACGTGCT-3’ and 5’-477
AACGATCTACGGCAGTACACCGTTG-3’ for scyl2b-3, and 5’-478
GAATTCACAGCAGAACATGTGCT-3’ and 5’-479
TCATAATAGATCCAATAGAGATGGTTGTGAACCA-3’ for scyl2b-4. To check whether or 480
not the transcripts of SCYL2A and SCYL2B are produced in scyl2a, scyl2b, and double 481
mutant alleles, RNA was extracted from the mutant alleles with a previously described 482
method (Onate-Sanchez and Vicente-Carbajosa, 2008), and was reverse-transcribed to 483
cDNA. Then, RT-PCR was conducted with the cDNA and the following primers: 5’-484
TCCTGGTCTCGCTTGGAAGCTTTA-3’ and 5’-TGCTGCTTTGAACCATTTGGCTTAG-3’ 485
for SCYL2A, 5’-CACCATGTCGATAAACATGAAAACATTTACTCAAGCTC-3’ and 5’-486
TCATAATAGATCCAATAGAGATGGTTGTGAACCA-3’ for SCYL2B. As a control gene, β-487
tubulin (TUB2, AT5G62690) was amplified with the primers 5'-488
GCCAATCCGGTGCTGGTAACA-3' and 5'- CATACCAGATCCAGTTCCTCCTCCC-3'. PCR 489
amplification was performed with 37 cycles and visualized using ethidium bromide after 490
separation on agarose gels. 491
492
Phylogenetic Analysis and Similarity Matrix 493
494
Alignment of protein sequences was performed with ClustalW in Mega4. A phylogenetic 495
tree was constructed using Neighbor-joining, Poisson correction, pairwise deletion, and 496
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24
bootstrap values with 10,000 replicates in Mega4. The similarity matrix was generated 497
using pairwise sequence comparison of 16 sequences to determine the p-distance, the 498
number of amino acid substitutions per site. Protein sequence identity is shown as (1 - p-499
distance)*100. 500
501
Root Hair Length Measurement and Root Hair Phenotype Analysis 502
503
Photographs of three-day old roots grown in the complete nutrient plates were taken using 504
a Nikon SMZ1500 microscope and a Q-Imaging Retiga cooled 12-bit camera. Root hair 505
length was measured by using NIH ImageJ software program in two separate regions of 506
roots: 2 mm region below hypocotyls and 2 mm region starting 0.5 mm above the root hair 507
differentiation zone. Fifteen root hairs per each root were measured avoiding root hairs 508
that were growing into the medium and ten roots per genotype were used for root hair 509
length measurements. Two independent experiments were performed and similar results 510
were obtained. The number of abnormal root hairs was counted based on five different 511
categories: normal shape, v shape, y shape, wavy shape, and bulged shape. About 70 to 512
100 root hairs per each seedling were counted in 5 mm region starting 0.5 mm above the 513
root hair differentiation zone and ten seedlings were used in the analysis. Data were 514
displayed as percentages of each root hair class in wild type and scyl2b-3. 515
516
Phenotype Assay 517
518
Photographs of ten-day old plants grown in the complete nutrient plates were taken for 519
primary root length measurement and subjected to shoot fresh weight measurement. For 520
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25
each mutant, ten seedlings per plate were grown, and six replicate plates were used. For 521
the measurement of primary root length, fifty roots per each mutant were used. Shoot fresh 522
weight was measured by bulking 10 plants from each of six plates. Statistical significance 523
was evaluated with a Student’s t test. Two independent experiments were performed and 524
similar results were obtained. 525
526
Quantitative Real-Time PCR analysis 527
528
Various tissues from seven-day-old seedlings or one-month-old plants were harvested and 529
total RNA was isolated as described previously (Onate-Sanchez and Vicente-Carbajosa, 530
2008). RNA was quantified and treated with RQ RNase-free DNase I (Promega). DNase-531
treated RNA was tested for genomic DNA contamination, and the quality of total RNA was 532
determined by agarose gel electrophoresis. Two and a half micrograms of DNA-free RNA 533
was then reverse transcribed using the First-Stand Synthesis System (Invitrogen). 534
Quantitative real-time RT-PCR analysis was performed using the StepOnePlus Real-Time 535
PCR system (Applied Biosystems) and Platinum SYBR Green qPCR SuperMix-UDG 536
(Invitrogen). To analyze the expression of SCYL2A and SCYL2B by real-time qPCR, 537
primers 5’-TCAAACCACAGTCCAGGAGGTTACAG-3’ and 5’-538
TCAAGCGCCTGAACAACATGAACC-3’ were used for SCYL2A gene and primers 5’- 539
AGTACAGGAAGTTACGGGACCAAAG-3’ and 5’- 540
AGCGGCTCCGTAACTAAAGCCATAGC-3’ were used for SCYL2B gene. For the 541
normalization of SCYL2A and SCYL2B transcripts, ACTIN7 gene (AT5G09810) was 542
amplified with the primers 5'-ACCACTACCGCAGAAC-3' and 5'-GCTCATACGGTCAGCA-543
3' and was used as an internal control. Statistical differences in SCYL2A and SCYL2B 544
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26
transcript levels between samples were evaluated by a Student’s t test using ΔΔCt values 545
(Yuan et al., 2006). Three biological replicates were used to generate means and statistical 546
significance. 547
548
Plasmid Construction and Plant Transformation 549
550
To generate the vector constructs used in this study, the open reading frame of full-length 551
genes was amplified by PCR using NEB Phusion polymerase and the following primers: 552
sense 5'-CACCATGTCGATTAATATGAGAACGCTAA-3' and antisense 5'-553
TCACAATAGATCTAACAAAGATGGC-3' for SCYL2A (AT1G22870), sense 5'-554
CACCATGTCGATTAATATGAGAACGCTAA-3' and antisense 5'-555
CAATAGATCTAACAAAGATGGC-3' for SCYL2A without a stop codon, sense 5'- 556
CACCATGTCGATAAACATGAAAACATTTACTCAAGCTC-3' and antisense 5'-557
TCATAATAGATCCAATAGAGATGGTTGTGAACCA-3' for SCYL2B (AT1G71410), sense 5'- 558
CACCATGTCGATAAACATGAAAACATTTACTCAAGCTC-3' and antisense 5'-559
TAATAGATCCAATAGAGATGGTTGTGAACCA-3' for SCYL2B without a stop codon, sense 560
5’- CACCATGGCGGCTGCTAACGCGCCCATCATTATG-3’ and antisense 5’-561
TTAGTAGCCGCCCATCGGTGGCATTCCA-3’ for CHC1 (AT3G11130), sense 5’-562
CACCATGAGTGACGTGTTTGATGGATAT-3’ and antisense 5’-563
TTACTTGGTGAGTTTGAAGTACAAGATGAT-3’ for VTI11 (AT5G39510), sense 5’-564
CACCATGAGCGACGTATTTGAAGGGTACGAGCGT-3’ and antisense 5’-565
TTAATGAGAAAGCTTGTATGAGATGATCAA-3’ for VTI12 (AT1G26670), sense 5’-566
CACCATGAATCCCTTTTCTTCT-3’ and antisense 5’-TCACAACCCGCGAGGGAA-3’ for 567
AP-1 (AT1G23900), sense 5’-CACCATGACCGGAATGAGAGGTCTCTCCGTA-3’ and 568
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27
antisense 5’-TTAAAGTAAGCCAGCGAGCATAGCTCC-3’ for AP-2 (AT5G22770), sense 569
5’-CACCATGTCGTCGTCTTCCACTTCTATAATG-3’ and antisense 5’-570
TCACAAGAGAAAATCTGGAATTATAAC-3’ for AP-3 (AT1G48760). The PCR products 571
were inserted into pENTR/D-TOPO following the manufacturer's instructions (Invitrogen). 572
The inserts were sequenced to make sure that no changes were introduced by PCR. The 573
SCYL2B gene in pENTR/D-TOPO was introduced into the destination plasmid 574
pEARLYGATE104:N-YFP or pEARLYGATE101:N-HA to yield pEARLYGATE:YFP(or 575
HA):SCYL2B, which allows the expression of fusion proteins with YFP or HA tag at the N-576
terminal position under control of the cauliflower mosaic virus 35S promoter. For the 577
colocalization study between SCYL2 proteins and CHC1 in tobacco leaf epidermal cells, 578
genes of SCYL2A, SCYL2B, and CHC1 in pENTR/D-TOPO were cloned into either 579
pMDC43:N-GFP vector or pB7WGR2:N-RFP to generate overexpression vectors with N-580
terminal fluorescent protein fusion genes. The SCYL2A/2B without stop codons were 581
cloned into pMDC83:C-GFP vector and pB7RWG2:C-RFP to yield C-terminal GFP (and 582
RFP)-fused SCYL2A/2B overexpression vectors. 583
To generate the pGreenII:SCYL2A/2Bpromoter:GUS plasmids, pGreenII:GUS 584
plasmid (Hellens et al., 2000) was first digested by NcoI and HindIII restriction enzymes. 585
Then, DNA fragments containing SCYL2A and SCYL2B promoter regions (about 1.5 kb) 586
were amplified by PCR using NEB Phusion polymerase and the following primers: sense 587
5'-ATTATAAGAACACTACCTTGATCCTTTGCAAGTATTGGA-3' and antisense 5'-588
TACAGGACGTAACACCATTTTTTTTTGTCAGAATTTAACTGTTTCTCCA-3' for SCYL2A 589
promoter, sense 5’-ATTATAAGAACACTAATTCATCTCTTTGGTCAGTTACGT-3’ and 590
antisense 5’-TACAGGACGTAACACCATTTCTGCTGGATCCAATTACTCC-3’ for SCYL2B 591
promoter. The digested plamid and PCR-amplified DNA fragments were recombined by 592
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28
using a commercially available In-Fusion multiple DNA assembly cloning method 593
(Clontech Laboratories) to yield pGreenII:SCYL2A/2Bpromoter:GUS plasmids. Transgenic 594
Arabidopsis plants carrying pEARLYGATE:YFP:SCYL2B and pGreenII:GUS plasmids 595
containing SCYL2A and SCYL2B promoters were generated by Agrobacterium-mediated 596
transformation and T2 or T3 transgenic lines were used in this study. 597
To generate the pMDC43:EXPA7promoter:N-GFP plasmids carrying various N-terminal GFP 598
fused genes which encode proteins with root hair tip localization, the 35S promoter of 599
pMDC43 vector was replaced with the 1.5 Kb promoter of EXPA7 by the recombination of 600
pMDC43 DNA fragments digested with HindIII and KpnI and PCR-amplified DNA 601
fragments using primers 5’-602
CGACGGCCAGTGCCAAAGCTTAATTAGGGTCCAAGGTTTGTTC-3’ and 5’-603
CATTTTTTCTACCGGTACCTCTAGCCTCTTTTTCTTTATTCTTA-3’. Then, the open 604
reading frame of full-length genes of ROP2, RABA4B, RHD2, CSLD3, SYP123, and PRP3 605
was PCR-amplified using the following primers: sense 5'-606
CCGGTACCGAATTCGATGGCGTCAAGGTTTATAAAGTGT-3' and antisense 5'-607
ATATCTCGAGTGCGGTCACAAGAACGCGCAACGGTTCTT-3' for ROP2 (AT1G20090), 608
sense 5’-CCGGTACCGAATTCGATGGCCGGAGGAGGCGGATAC-3’ and antisense 5’-609
ATATCTCGAGTGCGGTCAAGAAGAAGTACAACAAGTGCT-3’ for RABA4B (AT4G39990), 610
sense 5'-CCGGTACCGAATTCGATGTCTAGAGTGAGTTTTGAAGTG-3' and antisense 5'- 611
ATATCTCGAGTGCGGTTAGAAATTCTCTTTGTGGAAGGA-3' for RHD2 (AT5G51060), 612
sense 5’-CCGGTACCGAATTCGATGGCGTCTAATAATCATTTCATG-3’ and antisense 5’-613
ATATCTCGAGTGCGGTCATGGGAAAGTGAAAGATCCTCC-3’ for CSLD3 (AT3G03050), 614
sense 5'-CCGGTACCGAATTCGATGAACGATCTTATCTCAAGCTCA-3' and antisense 5'-615
ATATCTCGAGTGCGGTCAAGGTCGAAGTAGAGTGTTAAA-3' for SYP123 (AT4G03330), 616
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29
sense 5’-CCGGTACCGAATTCGATGGCGATCACACGCTCCTCCT-3’ and antisense 5’-617
ATATCTCGAGTGCGGTCAGTATTTGGGAGTGGCGGG-3’ for PRP3 (AT3G62680) and 618
recombined with pENTR2B (Invitrogen) DNA fragments amplified by PCR using primers 5’-619
CCGCACTCGAGATATCTAGAC-3’ and 5’-CGAATTCGGTACCGGATC-3’. The resulting 620
pENTR2B vectors with genes were recombined with the destination plasmid 621
pMDC43:EXPA7promoter plasmid to yield the GFP-fusion genes in pMDC43:EXPA7promoter 622
plasmids and T2 transgenic plants in the background of Col-0 and scyl2b mutants were 623
generated and used for the root hair tip localization study. 624
625
GUS Histochemical Assay 626
627
Staining for GUS activity was carried out as described previously (Harrison et al., 2006). 628
Briefly, about 2-week old plants grown on plates were dipped in GUS solution and 629
vacuum-infiltrated for 20 min. Samples were then incubated for 2 days at 37°C, cleared in 630
70% v/v ethanol and mounted in water prior to examination. GUS-stained tissues and 631
plants shown in this paper represent the typical results of at least two independent lines for 632
each construct. 633
634
Protein Interaction Assay 635
636
For BiFC assay, genes in pENTR/D-TOPO were introduced into the destination plasmid 637
pSITE-BiFC-C1nec or pSITE-BiFC-C1cec to yield nYFP- or cYFP- fusion constructs 638
(Martin et al., 2009). Then, tobacco leaves were infiltrated with agrobacterium carrying the 639
BiFC constructs as described previously (Sparkes et al., 2006). Three days after 640
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30
agrobacterium infiltration, YFP fluorescence images were obtained from tobacco leaf 641
epidermal cells. The expression level of nYFP- or cYFP- fusion proteins in tobacco leaf 642
epidermal cells was detected by western blotting using rabbit polyclonal Anti-GFP antibody 643
(ab290, abcam, UK). For co-immunoprecipitation assay, four week-old tobacco leaves 644
were co-infiltrated with agrobacterium carrying the HA- and YFP fusion genes. Three days 645
after infiltration, leaves were frozen, ground, and protein-extracted in the extraction buffer 646
(250 mM Suc, 20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM 647
EGTA, 1% Nonidet P-40, 0.1% Na-deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 648
EDTA-free protease inhibitor cocktail complete (Roche), and 10 mM NaF). The total 649
protein extracts were centrifuged at 10,000g for 10 min to remove cell debris and the 650
supernatant was incubated with anti-HA magnetic beads (Pierce) or GFP-Trap beads 651
(ChromoTek) for 2 h at 4°C. Beads were washed four times in the extraction buffer 652
containing 0.1% Nonidet P-40, then washed once with the same extraction buffer without 653
detergent. The protein beads were boiled in loading buffer and the beads were removed. A 654
quarter of the elutes and 10 g protein from total lysates (input fraction) were loaded and 655
run on a 4-12% ExpressPlus PAGE Gel (Genscript, USA) and were transferred with 100V 656
for 1 h by a wet transfer method to an Amersham Hybond-P PVDF membrane (GE 657
healthcare). Membranes was washed briefly in PBST buffer (10 mM phosphate buffer pH 658
7.2, 150 mM NaCl, 0.2% Tween-20) and blocked in PBST buffer with 1% BSA (Sigma) for 659
1 h. The membrane was incubated overnight at 4°C with either rabbit polyclonal Anti-GFP 660
antibody (1:7,500 dilution, ab290, abcam, UK) or rat monoclonal Anti-HA High affinity 661
antibody (1:7,500 dilution, Roche) in the PBST with 1% BSA, washed three times, and 662
incubated with either goat anti-rabbit IgG-HRP or goat anti-rat IgG-HRP (Santa Cruz 663
Biotechnology, USA, 1:7,500 dilution) in the PBST with 1% BSA at room temperature. After 664
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31
washing the membrane, signal was detected using the Western Bright Sirius HRP 665
Substrate (Advansta, USA) and a GE healthcare ImageQuant RT ECL Imager. 666
667
Fluorescent Organelle Markers and Microscopy 668
669
Monomeric RFP (mRFP):SCAMP1 (TGN marker) and mRFP:AtVSR2 (PVC marker) were 670
kindly provided from Liwen Jiang (Chinese University of Hong Kong). mRFP:SCAMP1 was 671
used as a marker for both TGN and PM since it localizes to these two compartments (Lam 672
et al., 2007). The other organelle markers used in this study were obtained from the 673
Arabidopsis stock centers (http://www.arabidopsis.org) and are also described on the web 674
site (http://www.bio.utk.edu/cellbiol/markers) and a paper published previously (Nelson et 675
al., 2007). For the colocalization studies, Arabidopsis leaf protoplasts were generated 676
using three week-old plants and transformed with plasmids by polyethylene glycol (PEG) 677
solution based on a previously described method (Yoo et al., 2007). Fluorescence signals 678
were observed 24 h to 48 h after the PEG transformation. Images were taken using a 679
Zeiss LSM 510 Meta confocal laser-scanning microscope equipped with C-Apochromat 680
40x N.A. 1.2 water immersion lens (Zeiss, Jena, Germany). For the colocalization studies 681
using YFP and CFP signals, 514 nm excitation wavelength with 535-590 nm emission filter 682
and 458 nm excitation wavelength with 480-520 nm emission filter were used, respectively. 683
For the colocalization studies using YFP and RFP signals, 488 nm excitation wavelength 684
with 500-550 nm emission filter and 543 nm excitation wavelength with 565-615 nm 685
emission filter were used, respectively. Images obtained by confocal microscope were 686
processed using NIH ImageJ software to remove low-level background fluorescence and 687
to enhance contrast for the increase of signal intensity. For colocalization images shown in 688
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32
Fig. 6, YFP images were rendered in green color and CFP or RFP images were rendered 689
in magenta color. Root hairs of transgenic Arabidopsis plants expressing YFP:SCYL2B 690
were observed under a Nikon Eclipse E800 widefield microscope and a 40×/1.3 NA oil-691
immersion lens with appropriate YFP filters and images were processed using imageJ 692
software. 693
694
Chemical Inhibitor and FM4-64 Uptake Assay 695
696
For chemical inhibitor assay, four week-old tobacco leaves were infiltrated with 697
agrobacterium carrying the 35S:GFP:SCYL2B fusion gene. Three days after infiltration, 698
tobacco leaf discs with 1 cm diameter were incubated in water containing either 50 µM 699
BFA or 30 µM wortmannin. Two hours after the incubation, GFP fluorescent images were 700
taken using a Zeiss LSM 700 confocal laser-scanning microscope (Zeiss, Jena, Germany). 701
Two independent experiments were performed and about five leaf discs are used for each 702
sample. For FM4-64 uptake assay, about seven day-old seedlings of Col-0 and scyl2a-3 703
scyl2b-3 double mutants grown on ammonium free complete nutrient agar medium (Jung 704
et al., 2009) were incubated with the same agar-free nutrient solution containing 2 M FM 705
4-64 dye for 5, 30, and 60 min. Then, fluorescent images were taken under a Zeiss LSM 706
700 confocal laser-scanning microscope using 488 nm excitation wavelength with 585-610 707
nm emission filter. Two independent experiments were performed and about five to ten 708
seedlings were observed for each sample. 709
710
Accession Numbers 711
712
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33
Sequence data from this article can be found in the Arabidopsis Genome Initiative or 713
GenBank/EMBL databases under the following accession numbers: SCYL2A 714
(AT1G22870), SCYL2B (AT1G71410), CHC1 (AT3G11130), VTI11 (AT5G39510), VTI12 715
(AT1G26670), ROP2 (AT1G20090), RABA4B (AT4G39990), RHD2 (AT5G51060), CSLD3 716
(AT3G03050), SYP123 (AT4G03330), PRP3 (AT3G62680), AP-1 (AT1G23900), AP-2 717
(AT5G22770), and AP-3 (AT1G48760) 718
719
Supplemental Data 720
721
The following materials are available in the online version of this article. 722
Supplemental Figure S1. The segmental duplication of SCYL2 genes in Arabidopsis. 723
Supplemental Figure S2. Tip localization of SCYL2B in growing root hairs.. 724
Supplemental Figure S3. SCYL2B is not localized at peroxisomes, mitochondria, plastids, 725
and PM. 726
Supplemental Figure S4. SCYL2B is localized at the Golgi, TGN, and PVC in Arabidopsis 727
leaf epidermal cells. 728
Supplemental Figure S5. Western blotting analysis to show the expression level of 729
nYFP:SCYL2B with cYFP, cYFP:CHC1, cYFP:VTI11, and cYFP:VTI12 in tobacco leaf 730
epidermal cells. 731
Supplemental Figure S6. Coimmunoprecipitation of SCYL2B with CHC1, VTI11, and 732
VTI12 using GFP trap beads. 733
Supplemental Figure S7. Full scans of immunoblots. 734
Supplemental Figure S8. CSLD3 is mislocalized in root hairs of scyl2b mutants. . 735
Supplemental Figure S9. A clathrin binding motif (SLLDLL) on SCYL2B does not affect 736
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34
the interaction between SCYL2B and CHC1. 737
Supplemental Figure S10. SCYL2B does not interact with adaptor proteins. 738
Supplemental Figure S11. Effect of brefeldin A (BFA) and wortmannin, endomembrane 739
trafficking inhibitors, on the localization of SCYL2B in tobacco leaf epidermal cells. 740
Supplemental Figure S12. Comparison of localization pattern of SCYL2B expressed by 741
various promoters in tobacco leaf epidermal cells. 742
Supplemental Figure S13. FM4-64 uptake assay in Col-0 and scyl2a-3 scyl2b-3 double 743
mutants. 744
Supplemental Figure S14. SCYL2 proteins function in the development of root columella 745
cells in Arabidopsis. 746
Supplemental Table S1. Percentages of YFP:SCYL2B-positive spots overlapped with 747
organelle marker proteins. 748
749
ACKNOWLEDGEMENTS 750
751
We thank Howard Berg (Donald Danforth Plant Science Center) for microscopy 752
assistance; Liwen Jiang for providing fluorescent organelle markers; Sandeep Tata for the 753
preparation of tobacco plants for BiFC assay; Yves Poirier for supporting experimental 754
works. Inhwan Hwang and Dong Wook Lee were supported by the Cooperative Research 755
program for Agriculture Science & Technology Development (Project No. 010953012016), 756
Rural Development Administration, Republic of Korea. Stephen Beungtae Ryu was 757
supported by grant (PJ01109103) from the Next-Generation BioGreen 21 Program (SSAC), 758
Korea Rural Development Administration. 759
760
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35
AUTHOR CONTRIBUTIONS 761
762
J.J., D.P.S., D.W.L., S.B.R., and I.H. conceived and designed the experiments. J.J. and 763
D.W.L. performed the experiments. J.J. and D.W.L. analysed the data., J.J., D.P.S., .S.B.R, 764
and I.H. wrote the manuscript. 765
766
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36
Figures and Legends 767
768
Figure 1. SCYL2 is conserved in eukaryotes. 769
A, A similarity matrix is shown to represent the percentage of amino acid identity between 770
species across the SCYL2 proteins. B, Neighbor-joining phylogenetic tree showing the 771
relationship between SCYL2 proteins from eukaryote species. The numbers above the 772
nodes are percentages of bootstrap confidence levels from 10,000 replicates. Branch 773
length represents the number of amino acid changes per site in accordance with the scale 774
bar. Species of SCYL2 proteins in (A) and (B) are Arabidopsis thaliana, Carica papaya, 775
Populus trichocarpa, Glycine max, Zea mays, Oryza sativa japonica, Homo sapiens, Pan 776
troglodytes, Canis lupus familiaris, Bos Taurus, Mus musculus, Rattus norvegicus, Gallus 777
gallus, Drosophila melanogaster, and Anopheles gambiae. 778
Figure 2. Gene structure of SCYL2A and SCYL2B and location of T-DNA insertions. 779
A, A schematic diagram shows the positions of T-DNA insertions in scyl2a and scyl2b 780
alleles. Black boxes represent exons. Lines between black boxes represent introns. White 781
boxes represent UTR regions. White triangles indicate T-DNA insertion sites. The position 782
of primers that are used in (C) to check the presence of full-length transcripts of SCYL2A 783
or SCYL2B is indicated by arrows. B, Salk and Sail lines used to isolate scyl2a and scyl2b 784
alleles and their T-DNA locations indicated. Four different double mutants were obtained 785
between scyl2a and scyl2b mutants. C, RT-PCR analysis of SCYL2A and SCYL2B 786
transcripts in wild type, single, and double mutants. The position of primers used is shown 787
in (A). A β-tubulin gene (TUB2) was used as a positive control for the RT-PCR. All scyl2a 788
and scyl2b alleles are knock-out mutants, except for scyl2b-2, which has a decreased level 789
of full-length transcripts. Single PCR reactions were performed with cDNA from individual 790
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37
plants of each genotype. 791
Figure 3. SCYL2B functions in root hair development. 792
A, Light microscope images showing root hair length of wild-type Col-0, scyl2a, scyl2b, 793
and scyl2a scyl2b double mutant alleles. Three-day-old roots were used for the 794
measurement of root hair length in (A) and (B). Scale = 0.5 mm. B, Root hair length of 795
seedlings of wild-type Col-0, scyl2a, scyl2b, and scyl2a scyl2b double mutant alleles. Root 796
hair length was measured in two separate regions of root: 2 mm region below hypocotyls 797
(left graph and middle panel) and 2 mm region starting 0.5 mm above the root hair 798
differentiation zone (right graph and middle panel). Fifteen root hairs per each seedling 799
were measured and ten seedlings per genotype were used for root hair length 800
measurements (n = 10 seedlings, means ± SE). C, Abnormal root hairs in scyl2b-3. Root 801
hairs in scyl2b-3 were classified into five classes based on their phenotype (normal, v 802
shape, y shape, wavy, and bulged). Representative images of each class are shown (Left). 803
Percentages of each root hair class were determined in WT plants and scyl2b-3 (Right). 804
About 70 to 100 root hairs per each seedling were counted in 5 mm regions starting 0.5 805
mm above the root hair differentiation zone (n = 10 seedlings, means ± SE). At least two 806
independent experiments were performed. 807
808
Figure 4. SCYL2A and SCYL2B are important in plant growth and development. 809
A, Images of ten-day-old wild-type Col-0, scyl2a-1, scyl2b-1, and scyl2a-1 scyl2b-1 double 810
mutants. Scale = 0.5 cm. B, Primary root length of the wild type and scyl2 knockouts. 811
Values are mean ± SE from 50 roots. C, Shoot fresh weights the wild type and scyl2 812
knockouts. Values are mean ± SE from 10 seedlings per replicate (n = 6 replicates). D, 813
Image of one month-old wild-type Col-0, scyl2a-1 scyl2b-1, and scyl2a-3 scyl2b-3. Scale = 814
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38
1 cm. 815
816
Figure 5. SCYL2A and SCYL2B expression levels in various tissues. 817
A, Quantitative real-time PCR analysis of SCYL2A and SCYL2B expression in various 818
tissues. Tissue from 7-day-old plants and 1 month-old plants were used in this analysis. 819
The levels of SCYL2A and SCYL2B transcripts were normalized to ACTIN7. Three 820
biological replicates were used and error bars represent SE. B, Spatial localization of 821
SCYL2A and SCYL2B. Histochemical localization of GUS activity in plants carrying 822
SCYL2Apromoter:GUS (Left) and SCYL2Bpromoter:GUS (Right). About 2 week-old plants grown 823
on plates were used for the visualization of GUS activity. Arrows indicate GUS staining. 824
Figure 6. SCYL2B is localized at the Golgi, TGN, and PVC . 825
A, YFP:SCYL2B is tip localized in growing root hairs. B, The tip localization pattern 826
disappears in mature root hairs. Root hairs of transgenic Arabidopsis plants expressing 827
YFP:SCYL2B were observed under wide-field fluorescent microsope. Scale = 10 μm. C to 828
E, Localization of SCYL2B in various subcellular organelles. Arabidopsis leaf protoplasts 829
were co-transformed with YFP:SCYL2B and indicated constructs which encode protein 830
markers for the Golgi (C), TGN (D), and PVC (E). The localization of SCYL2B was 831
examined by the detection of YFP, CFP, and RFP signal under confocal microscope. 832
White color indicates strong colocalization. Arrows indicate some spots showing 833
colocalization. Scale = 20 μm. 834
835
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39
836
Figure 7. SCYL2B interacts with CHC1, VTI11, and VTI12. 837
A, BiFC assay showing in vivo interactions of SCYL2B with CHC1, VTI11, and VTI12. 838
nYFP:SCYL2B construct was transiently co-expressed with cYFP fusion constructs such 839
as CHC1, VTI11, and VTI12 in tobacco leaf epidermal cells via agrobacterium infiltration. 840
Bright field images and YFP fluorescence images are shown. Scale = 10 μm. B, 841
Coimmunoprecipitation of SCYL2B with CHC1, VTI11, and VTI12. YFP and YFP:SCYL2B 842
constructs were transiently co-expressed with HA:CHC1, HA:VTI11, and HA:VTI12 in 843
tobacco leaf epidermal cells. HA-tagged fusion proteins were co-immunoprecipitated by 844
anti-HA antibody-conjugated magnetic beads and probed with anti-GFP antibody. 845
IP,immunoprecipitation; Input, total protein extracts before IP; Bound, bound fraction after 846
IP. 847
Figure 8. SCYL2B colocalizes with SCYL2A and CHC1. 848
A to D, Colocalization of SCYL2B with SCYL2A and CHC1. Tobacco leaf epidermal cells 849
were co-infiltrated with Agrobacterium strains carrying constructs: GFP:SCYL2B and 850
SCYL2B:RFP (A), GFP:SCYL2B and SCYL2A:RFP (B), GFP:SCYL2B and RFP:CHC1 (C), 851
and GFP:SCYL2A and RFP:CHC1 (D). The localization of fluorescent proteins was 852
examined by the detection of GFP and RFP signal under confocal microscope. 853
Representative images of each sample are shown. White color indicates strong 854
colocalization. Arrows indicate some spots showing colocalization. Scale = 10 μm. 855
856
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40
857
Figure 9. CSLD3 is mislocalized in root hairs of scyl2b mutants. . 858
Localization of proteins which are known to function at the root hair tip was observed in the 859
background of Col-0 and scyl2b-3 mutants. The expression of N-terminal GFP fusion 860
proteins of ROP2, RHD2, RABA4B, SYP123, PRP3, and CSLD3 was driven by EXPA7 861
promoter, a root hair specific promoter. For each gene construct, at least 5 independent T2 862
transgenic plants were used in two independent experiments that yielded similar results. 863
Root hairs of transgenic plants were observed under confocal microsope. A white arrow 864
indicates the difference of GFP:CSLD3 localization. Scale = 10 μm. 865
866
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41
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Figure 1. SCYL2 is conserved in eukaryotes.
A, A similarity matrix is shown to represent the percentage of amino acid identity between
species across the SCYL2 proteins. B, Neighbor-joining phylogenetic tree showing the
relationship between SCYL2 proteins from eukaryote species. The numbers above the nodes
are percentages of bootstrap confidence levels from 10,000 replicates. Branch length
represents the number of amino acid changes per site in accordance with the scale bar.
Species of SCYL2 proteins in (A) and (B) are Arabidopsis thaliana, Carica papaya, Populus
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trichocarpa, Glycine max, Zea mays, Oryza sativa japonica, Homo sapiens, Pan troglodytes,
Canis lupus familiaris, Bos Taurus, Mus musculus, Rattus norvegicus, Gallus gallus, Drosophila
melanogaster, and Anopheles gambiae.
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Figure 2. Gene structure of SCYL2A and SCYL2B and location of T-DNA insertions.
A, A schematic diagram shows the positions of T-DNA insertions in scyl2a and scyl2b alleles.
Black boxes represent exons. Lines between black boxes represent introns. White boxes
represent UTR regions. White triangles indicate T-DNA insertion sites. The position of primers
that are used in (C) to check the presence of full-length transcripts of SCYL2A or SCYL2B is
indicated by arrows. B, Salk and Sail lines used to isolate scyl2a and scyl2b alleles and their T-
DNA locations indicated. Four different double mutants were obtained between scyl2a and
scyl2b mutants. C, RT-PCR analysis of SCYL2A and SCYL2B transcripts in wild type, single,
and double mutants. The position of primers used is shown in (A). A β-tubulin gene (TUB2) was
used as a positive control for the RT-PCR. All scyl2a and scyl2b alleles are knock-out mutants,
except for scyl2b-2, which has a decreased level of full-length transcripts. Single PCR reactions
were performed with cDNA from individual plants of each genotype.
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Figure 3. SCYL2B functions in root hair development.
A, Light microscope images showing root hair length of wild-type Col-0, scyl2a, scyl2b, and
scyl2a scyl2b double mutant alleles. Three-day-old roots were used for the measurement of root
hair length in (A) and (B). Scale = 0.5 mm. B, Root hair length of seedlings of wild-type Col-0,
scyl2a, scyl2b, and scyl2a scyl2b double mutant alleles. Root hair length was measured in two
separate regions of root: 2 mm region below hypocotyls (left graph and middle panel) and 2 mm
region starting 0.5 mm above the root hair differentiation zone (right graph and middle panel).
Fifteen root hairs per each seedling were measured and ten seedlings per genotype were used
for root hair length measurements (n = 10 seedlings, means ± SE). C, Abnormal root hairs in
scyl2b-3. Root hairs in scyl2b-3 were classified into five classes based on their phenotype
(normal, v shape, y shape, wavy, and bulged). Representative images of each class are shown
(Left). Percentages of each root hair class were determined in WT plants and scyl2b-3 (Right).
About 70 to 100 root hairs per each seedling were counted in 5 mm regions starting 0.5 mm
above the root hair differentiation zone (n = 10 seedlings, means ± SE). At least two
independent experiments were performed.
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Figure 4. SCYL2A and SCYL2B are important in plant growth and development.
A, Images of ten-day-old wild-type Col-0, scyl2a-1, scyl2b-1, and scyl2a-1 scyl2b-1 double
mutants. Scale = 0.5 cm. B, Primary root length of the wild type and scyl2 knockouts. Values
are mean ± SE from 50 roots. C, Shoot fresh weights the wild type and scyl2 knockouts. Values
are mean ± SE from 10 seedlings per replicate (n = 6 replicates). D, Image of one month-old
wild-type Col-0, scyl2a-1 scyl2b-1, and scyl2a-3 scyl2b-3. Scale = 1 cm.
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Figure 5. SCYL2A and SCYL2B expression levels in various tissues.
A, Quantitative real-time PCR analysis of SCYL2A and SCYL2B expression in various tissues.
Tissue from 7-day-old plants and 1 month-old plants were used in this analysis. The levels of
SCYL2A and SCYL2B transcripts were normalized to ACTIN7. Three biological replicates were
used and error bars represent SE. B, Spatial localization of SCYL2A and SCYL2B.
Histochemical localization of GUS activity in plants carrying SCYL2Apromoter:GUS (Left) and
SCYL2Bpromoter:GUS (Right). About 2 week-old plants grown on plates were used for the
visualization of GUS activity. Arrows indicate GUS staining.
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Figure 6. SCYL2B is localized at the Golgi, TGN, and PVC .
A, YFP:SCYL2B is tip localized in growing root hairs. B, The tip localization pattern disappears
in mature root hairs. Root hairs of transgenic Arabidopsis plants expressing YFP:SCYL2B were
observed under wide-field fluorescent microsope. Scale = 10 μm. C to E, Localization of
SCYL2B in various subcellular organelles. Arabidopsis leaf protoplasts were co-transformed
with YFP:SCYL2B and indicated constructs which encode protein markers for the Golgi (C),
TGN (D), and PVC (E). The localization of SCYL2B was examined by the detection of YFP,
CFP, and RFP signal under confocal microscope. White color indicates strong colocalization.
Arrows indicate some spots showing colocalization. Scale = 20 μm.
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Figure 7. SCYL2B interacts with CHC1, VTI11, and VTI12.
A, BiFC assay showing in vivo interactions of SCYL2B with CHC1, VTI11, and VTI12.
nYFP:SCYL2B construct was transiently co-expressed with cYFP fusion constructs such as
CHC1, VTI11, and VTI12 in tobacco leaf epidermal cells via agrobacterium infiltration. Bright
field images and YFP fluorescence images are shown. Scale = 10 μm. B,
Coimmunoprecipitation of SCYL2B with CHC1, VTI11, and VTI12. YFP and YFP:SCYL2B
constructs were transiently co-expressed with HA:CHC1, HA:VTI11, and HA:VTI12 in tobacco
leaf epidermal cells. HA-tagged fusion proteins were co-immunoprecipitated by anti-HA
antibody-conjugated magnetic beads and probed with anti-GFP antibody.
IP,immunoprecipitation; Input, total protein extracts before IP; Bound, bound fraction after IP.
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Figure 8. SCYL2B colocalizes with SCYL2A and CHC1.
A to D, Colocalization of SCYL2B with SCYL2A and CHC1. Tobacco leaf epidermal cells were
co-infiltrated with Agrobacterium strains carrying constructs: GFP:SCYL2B and SCYL2B:RFP
(A), GFP:SCYL2B and SCYL2A:RFP (B), GFP:SCYL2B and RFP:CHC1 (C), and GFP:SCYL2A
and RFP:CHC1 (D). The localization of fluorescent proteins was examined by the detection of
GFP and RFP signal under confocal microscope. Representative images of each sample are
shown. White color indicates strong colocalization. Arrows indicate some spots showing
colocalization. Scale = 10 μm.
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Figure 9. CSLD3 is mislocalized in root hairs of scyl2b mutants. .
Localization of proteins which are known to function at the root hair tip was observed in the
background of Col-0 and scyl2b-3 mutants. The expression of N-terminal GFP fusion proteins of
ROP2, RHD2, RABA4B, SYP123, PRP3, and CSLD3 was driven by EXPA7 promoter, a root
hair specific promoter. For each gene construct, at least 5 independent T2 transgenic plants
were used in two independent experiments that yielded similar results. Root hairs of transgenic
plants were observed under confocal microsope. A white arrow indicates the difference of
GFP:CSLD3 localization. Scale = 10 μm.
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