identiﬁcation and characterization of arabidopsis …...identiﬁcation and characterization of...

Identification and Characterization of Arabidopsis SeedCoat Mucilage Proteins1[OPEN]

Allen Yi-Lun Tsai 2, Tadashi Kunieda3, Jason Rogalski, Leonard J. Foster, Brian E. Ellis, andGeorge W. Haughn*

Department of Botany (A.Y.-L.T., T.K., G.W.H.), Michael Smith Laboratories (A.Y.-L.T., J.R., L.J.F., B.E.E.), andDepartment of Biochemistry and Molecular Biology (L.J.F.), University of British Columbia, Vancouver, BritishColumbia, Canada V6T 1Z4

ORCID ID: 0000-0001-8164-8826 (G.W.H.).

Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins aredifficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterizationof proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrixcomposed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilageinclude those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutantseeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherentand adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are presentexclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTAABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcriptsand promoter activities were detected in developing seed coats, and their expression requires seed coat differentiationregulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed toidentify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be veryuseful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as amodel for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specificfeatures.

The plant cell wall plays key roles in structural sup-port, cell-cell cohesion, and interaction of the cell withthe environment. It is a dynamic structure and can bestrengthened or loosened in response to environmentalor developmental cues (Fry, 2000; Passardi et al., 2004).

Plant cell walls typically contain cellulose and hemi-cellulose and may include pectin or lignin dependingon the type of wall. In addition to these carbohydratecomponents, 5% to 10% of the cell wall biomass consistsof proteins (Cassab and Varner, 1988; Burton et al.,2010). Despite being a relatively minor component interms of cell wall biomass, these proteins are criticalregulators of the cell wall architecture and, therefore, itsphysical properties. For example, structural proteinscan cross-link various cell wall polysaccharides(Showalter, 1993), while carbohydrate-active enzymesmodify polysaccharide structure.

Since cell wall proteins are generally difficult to ex-tract and analyze, they remain a relatively poorly un-derstood component of the cell wall. Several factorscomplicate the analysis of cell wall proteins. First, theyoften undergo extensive posttranslational modifica-tions, such as Pro hydroxylation, glycosylation, and theaddition of GPI anchors (Jamet et al., 2008b; Albenneet al., 2013). These modifications not only alter proteinmass, thereby complicating protein identification, butthey also can anchor the proteins in the apoplast bycovalent or noncovalent interactions (Kieliszewski andLamport, 1994; Spiro, 2002), which make cell wallprotein extraction and identification more challenging.The extraction of cell wall proteins typically requiresharsh conditions (Lee et al., 2004; Jamet et al., 2008b)

1 This work was supported by the National Sciences and Engineer-ing Research Council (NSERC; Discovery Grants to B.E.E. and G.W.H.), a British Columbia Proteomic Network Graduate/PostdoctoralTraining Grant (to A.Y.-L.T.), the NSERC Collaborative Research andTraining Experience ProgramWorking onWalls (to A.Y.-L.T., G.W.H.,and B.E.E.), and the Japan Society for the Promotion of Science (Post-doctoral Fellowship for Research Abroad to T.K.).

2 Present address: Graduate School of Science and Technology,Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan.

3 Present address: Department of Biology, Faculty of Science andEngineering, Konan University, 8-9-1 Okamoto, Higashinada-ku,Kobe 658-8501, Japan.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:George W. Haughn ([email protected]).

A.Y.-L.T. designed the research; A.Y.-L.T., T.K., and J.R. per-formed the research; A.Y.-L.T., L.J.F., B.E.E., and G.W.H. analyzedthe data and wrote the article.

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that often lead to protein degradation and contamina-tion with cytoplasmic proteins, with a resulting de-crease in the quality of proteomic data. In addition, thecell wall resides in extracellular space and abuts theperimeters of adjacent cells. Since a variety of cell typeswith distinctive cell walls are found in most tissues andorgans, it is common that cell wall extracts typicallyinclude carbohydrate and proteins derived from mul-tiple cell types, and the relative contribution of specificcell types is difficult to assess. Despite these problems,several studies have characterized cell wall proteomesfrom different tissue types in various plant species, in-cluding the model plant Arabidopsis (Arabidopsis thali-ana; for review, see Albenne et al., 2013). Out of the;5,000 Arabidopsis genes that encode a predicted sig-nal peptide to allow a protein to enter the secretorypathway, 1,000 to 2,000 are thought to be cell wallproteins (Jamet et al., 2006). However, currently, mostpublished cell wall proteomes contain less than100 proteins each and are contaminated by cytoplasmicproteins to a variable extent, depending on the tissuetype and extraction techniques (Albenne et al., 2013).This suggests that many cell wall proteins remain to bediscovered and characterized and emphasizes the needfor better models and more robust methodologies.

Arabidopsis seed coat mucilage is a specialized layerof the extracellular matrix composed of cell wall car-bohydrates arranged in a distinct structure (for review,see Arsovski et al., 2010; Haughn and Western, 2012;Western, 2012; North et al., 2014, Voiniciuc et al., 2015c)that is used as a model to study cell wall structure andfunction. It contains cellulose and hemicellulose(Macquet et al., 2007a; Young et al., 2008; Harpaz-Saadet al., 2011; Mendu et al., 2011; Sullivan et al., 2011;Griffiths et al., 2014; Yu et al., 2014; Voiniciuc et al.,2015a, 2015b; Hu et al., 2016a, 2016b) but is particularlyrich in pectin, with unsubstituted rhamnogalacturonanI making up ;85% of the total mucilage carbohydrate(Western et al., 2000, 2001; Willats et al., 2001; Deanet al., 2007; Macquet et al., 2007a; Young et al., 2008).Similar to cell walls, Arabidopsis seed coat mucilagealso contains proteins. Forward and reverse geneticsstudies have identified several loci required for propermucilage synthesis, secretion, and extrusion (for re-view, see Haughn and Chaudhury, 2005; Arsovskiet al., 2010; Haughn and Western, 2012; Western, 2012;North et al., 2014; Francoz et al., 2015). Several of thesegene products are believed to be secreted to the muci-lage pocket or adjacent primary wall in the developingseed coat. For example, the mucilage-modifying en-zyme MUCILAGE MODIFIED2 (MUM2) is secreted tothe mucilage pocket during mucilage synthesis(Western et al., 2001; Dean et al., 2007; Macquet et al.,2007b). PEROXIDASE36 (PER36) has been shown tolocalize to the radial and tangential primary cell walladjacent to the mucilage pocket (Kunieda et al., 2013).Two other genes that encode proteins needed for normalmucilage, SUBTILISIN-LIKE SERINE PROTEASE1.7(SBT1.7; Rautengarten et al., 2008) and arabinofur-anosidase b-XYLOSIDASE1 (BXL1; Arsovski et al., 2009),

contain signal peptides and modify mucilage carbohy-drates. However, a thorough analysis of mucilage pro-teins has not been described.

The deposition of seed coat mucilage is known asmyxospermy and is common in angiosperms (Youngand Evans, 1973; Grubert, 1974). During differentiation,Arabidopsis seed coat epidermal cells synthesize mu-cilage components and deposit them between theplasma membrane and the primary wall at the junctionbetween the radial and tangential cell walls, forming aring-shaped mucilage pocket surrounding a volcano-shaped cytoplasmic column (Western et al., 2000;Windsor et al., 2000). A cellulose-rich secondary cellwall, the columella, is subsequently deposited beneaththe mucilage, gradually replacing the cytoplasm(Western et al., 2000; Windsor et al., 2000). Upon ex-posure of mature seeds to water, the pectin-rich muci-lage swells rapidly, ruptures the primary wall, andextrudes to encapsulate the seed. The extruded Arabi-dopsis seed mucilage has at least two distinct layers,nonadherent and adherent (Western et al., 2000;Macquet et al., 2007a). The outermost layer (non-adherent layer) is amorphous in appearance, composedprimarily of pectin and, as its name suggests, easilyseparated from the seed by gentle shaking. The layer ofmucilage adjacent to the seed coat has a distinct ray-likestructure, has cellulose and hemicellulose in addition topectin, and is strongly adherent to the seed surface.Relative to cell wall preparations from most other tissuetypes, seed coat mucilage can be easily extracted in largeamounts without contamination with cell wall materialfrom other cell types (Haughn and Chaudhury, 2005;Haughn and Western, 2012; North et al., 2014). Theseadvantages suggest that seed coatmucilage can yield cellwall proteomes that are potentially of higher qualitythan cell wall proteomes derived from other tissues.Here, we describe the extraction and proteomic analysisof the mature Arabidopsis seed coat mucilage and dis-cuss the protein profiles of the mucilage in comparisonwith other cell wall proteomes. In addition, we charac-terize a family of unknown proteins that are particularlyabundant in seed coat mucilage and strongly expressedin the developing seed coat.

RESULTS

Proteins Are a Component of Mucilage Extracted fromArabidopsis Seeds

In order to identify and characterize proteins integralto the seed coat mucilage, a protocol was developed toextract seed coat mucilage for protein analyses (Fig. 1).Upon hydration, Arabidopsis seed coat epidermal cellsextrude mucilage as two distinct layers: an outer non-adherent layer that detaches easily from the hydratedseed and a dense halo-like adherent layer that is boundtightly to the seed coat (Fig. 1A; Western et al., 2000).We took advantage of the different physical propertiesof these two layers to separate them by sequential ex-traction (Fig. 1B). Seeds imbibed in water were shaken

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gently to separate the nonadherent mucilage. The seedswere then shaken at high speed for several hours toremove the adherent mucilage (Fig. 1B). RutheniumRed and Calcofluor White staining showed that thepectin and cellulosic components of seed coat mucilagewere almost completely removed by the sequentialextraction (Fig. 1B; Supplemental Fig. S1). The har-vested adherent and nonadherent mucilage sampleswere chemically deglycosylated, trypsin digested, andanalyzed by MS to identify mucilage-associated pro-teins in these samples (Fig. 1B; Supplemental Data SetsS1 and S2). Of those proteins detected by this process,only the ones identified in more than one biologicalreplicate withMASCOT scores greater than 40 (where ascore of 25 or greater corresponds to a 5% false dis-covery rate), and at least once with multiple peptides,were considered for further analyses. Based on thesecriteria, 30 proteins were considered to be robustlyidentified from mucilage, all containing predicted sig-nal peptides (Supplemental Data Set S3). Cruciferin A1and cruciferin C were discarded from further analyses,since they are not known to be secreted to the apoplast.This leaves a total 28 potential mucilage proteins

identified (Table I; Supplemental Data Set S3). Oneprotein was found only in the nonadherent layer,15 only in the adherent layer, and the remaining12 proteins were found in bothmucilage layers (Table I).

Mucilage Proteins Are Functionally Similar to Other CellWall Proteins

When proteins found in each mucilage layer weresorted by their predicted functions (Table I; Fig. 2A),most fell within the various functional categories of cellwall proteins as defined by Albenne et al. (2013). Thesecategories include carbohydrate-active enzymes, oxi-doreductases, proteases, proteins involved in lipidmetabolism, arabinogalactan proteins, as well as mis-cellaneous proteins and proteins with unknown func-tions. The fact that seed coat mucilage-associatedproteins appear to be functionally similar to proteinsfrom other Arabidopsis cell wall proteomes reinforcesthe concept that seed coat mucilage is a specialized typeof cell wall (Haughn and Western, 2012). On the otherhand, nearly half of the specific mucilage-associated

Figure 1. Strategy to isolate and identify seedcoat mucilage proteins. A, Columbia-0 (Col-0)seed coat mucilage stained with RutheniumRed. Double-headed arrows depict the twomucilage layers. Bar = 100 mm. B, Schematicdepiction of the extraction and identification ofmucilage proteins. The nonadherent mucilageand adherent mucilage were extracted se-quentially. Proteins in each mucilage layerwere identified bymass spectrometry (MS) afterchemical deglycosylation and trypsin diges-tion. ddH2O, Distilled, deionized water.

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Identification of Seed Coat Mucilage Proteins


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protein isoforms were unique to mucilage and notidentified in other cell wall proteomes to date (Fig. 2B),including a family of unknown proteins (discussedbelow), RmlC-like cupin superfamily proteins, andGDSL lipases (Table I). Homologs of the RmlC-likecupin superfamily proteins and GDSL lipases arecommonly found in other cell walls (Albenne et al.,2013), suggesting that these proteins may representmucilage-specific isoforms.

In general, the adherent layer displays a richer andmore diverse protein profile compared with the non-adherent layer, including 15 proteins that are unique to

the adherent layer (Table I). Interestingly, this adherent-specific group includes a number of proteins involved inlipid metabolism (Fig. 2A). Otherwise, the numbers ofproteins that belong to each predicted functional class aremore or less comparable between the twomucilage layers(Fig. 2A). This suggests that the types of protein-mediatedbiological processes associated with mucilage modifica-tion in the apoplast are comparable within the two layers.

Since the seeds from which mucilage was obtained inthese experiments had not been processed in any wayprior to hydration and mucilage extraction, the possi-bility remains that the proteins we identified originate

Table I. Total proteins identified in mature Col-0 seed coat mucilage

Accession No. ATG No. Name Location Function WallProtDB

O04575 At1g62080 TESTA ABUNDANT (TBA3) Both Unknown Not detectedQ39168 At1g62000 TESTA ABUNDANT (TBA1) Both Unknown Not detectedO04573 At1g62060 TESTA ABUNDANT (TBA2) Both Unknown Not detectedQ9M8X3 At3g04170 RmlC-like cupin superfamily

proteinAdherent Oxidoreductase Not detected

Q9FK75 At5g45670 GDSL motif esterase/acyltransferase/lipase

Adherent Lipid metabolism Not detected

Q9FGY1 At5g49360 b-XYLOSIDASE1 (BXL1) Adherent Carbohydrate-active enzyme Multiple tissuesQ9LU14 At3g16370 GDSL motif esterase/

acyltransferase/lipaseAdherent Lipid metabolism Leaves

O65351 At5g67360 SUBTILISIN-LIKE SERINEPROTEASE1.7 (Sbt1.7, ARA12)

Both Protease Multiple tissues

Q9LV33 At3g18080 B-S GLUCOSIDASE44 (BGLU44) Both Carbohydrate-active enzyme Multiple tissuesQ9SD46 At3g50990 PEROXIDASE36 (PER36) Both Oxidoreductase HypocotylQ94CH6 At1g75900 GDSL motif esterase/

acyltransferase/lipaseAdherent Lipid metabolism Not detected

Q9FFN4 At5g63800 MUCILAGE MODIFIED2 (MUM2) Both Carbohydrate-active enzyme Multiple tissuesQ8L7S6 At1g65590 b-HEXOSAMINIDASE3 (HEXO3) Both Carbohydrate-active enzyme Multiple tissuesQ9SCV4 At2g28470 b-GALACTOSIDASE8 (BGAL8) Both Carbohydrate-active enzyme LeavesQ9SUS0 At4g23560 GLYCOSYL HYDROLASE9B15

(GH9B15)Adherent Carbohydrate-active enzyme Not detected

Q9M8X6 At3g04200 RmlC-like cupin superfamilyprotein

Both Oxidoreductase Not detected

Q9LLR6 At5g59310 LIPID TRANSFER PROTEIN4(LTP4)


Q9LS40 At3g18490 ASPARTIC PROTEASE IN GUARDCELL1 (ASPG1)

Both Protease Multiple tissues

Q8VY93 At4g26790 GDSL motif esterase/acyltransferase/lipase


Q9FMK9 At5g63140 PURPLE ACID PHOSPHATASE29(PAP29)

Adherent Miscellaneous Roots

Q9LDB4 At3g08770 LIPID TRANSFER PROTEIN6(LTP6)

Adherent Lipid metabolism Roots

Q9LEY1 At5g08260 SERINE CARBOXYPEPTIDASE-LIKE35 (SCPL35)

Both Protease Not detected

Q94BT2 At3g07390 AUXIN-INDUCED IN ROOTCULTURES12 (AIR12)

Adherent Oxidoreductase Multiple tissues

Q9LZX4 At3g60900 FASCICLIN-LIKEARABINOGALACTANPROTEIN10 (FLA10)

Adherent Arabinogalactan protein Cell culture

Q9LHF1 At3g24480 Leu-rich repeat family protein Adherent Structural Multiple tissuesP43297 At1g47128 RESPONSIVE TO

DEHYDRATION21A (RD21a)Adherent Protease Multiple tissues

Q9SVU5 At4g28780 GDSL motif esterase/acyltransferase/lipase


Q66GR0 At5g06390 FASCICLIN-LIKEARABINOGALACTANPROTEIN17 (FLA17)

Nonadherent Arabinogalactan protein Not detected


Tsai et al.



from sources other than themucilage. In order to addressthis concern, the nonadherent mucilage protein profilesobtained from Col-0 seeds were compared with proteinprofiles obtained from the seed surface extracts from themucilage mutants mum2-10 and apetala 2-7 (ap2-7; Fig.3A). mum2-10 seeds synthesize mucilage but do not ex-trude it when hydrated (Dean et al., 2007; Macquet et al.,2007b), whereas ap2-7 seed coat epidermal cells fail todifferentiate and, therefore, do not synthesize mucilage(Jofuku et al., 1994; Western et al., 2001; Dean et al.,2011). Since mucilage can only be extracted from hy-drated Col-0 seeds, proteins that are significantly over-represented in Col-0 nonadherent mucilage comparedwith mum2-10 and/or ap2-7 seed surface extracts wouldbe predicted to be derived from the extruded mucilage.Several mucilage proteins identified were indeed foundat much higher levels in Col-0 compared with mum2-10and ap2-7 (Fig. 3B; Supplemental Data Sets S4 and S5).Overall, the recovery ofmucilage-associated proteinwasreduced by;90%whenmum2-10 seed was used and by

;99% when ap2-7 seed was used compared with Col-0seed (Fig. 3B). These data support the hypothesis thatproteins identified in this study are derived from ex-truded mucilage of seed coat epidermal cells and notfrom the primary wall.

The Identity of Many Mucilage Proteins Is Consistent witha Role in Mucilage/Cell Wall Modification

The collection of enzymes identified by our pro-teomics analyses includes all the secreted enzymesrequired for normal mucilage extrusion that havebeen identified previously by genetic analysis:MUM2 (At5g63800/Q9FFN4; Dean et al., 2007), BXL1(At5g49360/Q9FGY1; Arsovski et al., 2009), PER36(At3g50990/Q9SD46; Kunieda et al., 2013), and SBT1.7(At5g67360/O65351; Rautengarten et al., 2008; Table I).Their identification here thus validates the robustnessof the proteomic analysis.

Figure 2. Mucilage proteins are functionallysimilar to other cell wall proteins. A, Numbers ofseed coat mucilage proteins from each mucilagelayer sorted by the cell wall protein functionalcategories. B, Proportions of mucilage proteinspreviously identified in cell walls from other tissuetypes as documented by WallProtDB. Numbersdenote the number of proteins in each category,while percentages denote the proportion of pro-teins that occupy each category.






In an attempt to determine the roles of othermucilageproteinswe identified, plant lineswith T-DNA insertionsin the genes b-GLUCOSIDASE44 (At3g18080/Q9LV33),b-HEXOSAMINIDASE3 (HEXO3; At1g65590/Q8L7S6),ASPARTIC PROTEASE IN GUARD CELL1 (ASPG1;At3g18490/Q9LS40), AUXIN INDUCIBLE IN ROOTS12(At3g07390/Q94BT2),RESPONSIVETODEHYDRATION1a(RD21a; At1g47128/P43297), and SERINE CARBOXY-PEPTIDASE-LIKE35 (At5g08260/Q9LEY1) were char-acterized (Supplemental Fig. S2; Supplemental Table S1).These genes were chosen because they do not appear tohave homologs that are also expressed in seed coat epi-dermal cells, thus decreasing the possibility of functionalredundancy obscuring mutant phenotypes. In each case,the T-DNA insertion decreased or eliminated thesteady-state levels of transcript in homozygous lines(Supplemental Fig. S2). Seeds of each insertional mutantwere imbibed inwater, 0.05 M EDTA, 0.05 MCaCl2, or 0.5 M

Na2CO3, stained with Ruthenium Red, and examined forseed mucilage abnormalities. EDTA is believed to loosenmucilage bydisrupting the homogalacturonan salt bridgesthrough Ca2+ chelation (Western et al., 2001; Rautengartenet al., 2008; Saez-Aguayo et al., 2013; Voiniciuc et al., 2013).Na2CO3 treatment also loosens mucilage, possibly bycleaving cross-linking ester bonds between homo-galacturonan polymers (Selvendran and Ryden, 1990; Fry,2000; McCartney and Knox, 2002). In contrast, mucilageextruded in a CaCl2 solution is more compact and stainsmore intensely with Ruthenium Red than mucilageextruded in water, presumably by enhancing Ca2+ saltbridging betweenmucilage homogalacturonanmolecules.However, no clear mucilage defects were found in anymucilage protein mutant lines under the conditions tested(Supplemental Fig. S3), suggesting that if the correspond-inggeneproducts have a role inmucilagemodification, themutant phenotypemust be relatively subtle or conditional.

Figure 3. Proteins identified aregenuinely associated with mucilage.A, Schematic depiction of the mu-cilage protein quantification inmum2-10 and ap2-7 seed surfaceextracts relative to Col-0 non-adherent mucilage. ddH2O, Dis-tilled, deionized water. B, Relativelevels of mucilage proteins in Col-0nonadherent mucilage, mum2-10seed surface extract, and ap2-7 seedsurface extract. Values are normal-ized to Col-0. Averages 6 SD areshown; n = 3. *, P , 0.01; and **,P , 0.001.


Tsai et al.







TBA Proteins Were Found to Be Highly Abundant in SeedCoat Mucilage

Among themucilage proteins identified, three proteins ofthe unknown protein family 0540 (UPF0540), At1g62000/Q39168,At1g62060/O04573, andAt1g62080/O04575,wereof particular interest because they were consistently identi-fied as the most abundant proteins in almost all samples(Table I; Supplemental Data Set S4). Consistent with theseprotein data, the corresponding genes were found to beexpressed in the seed coat at very high levels (SupplementalFig. S4; Schmid et al., 2005;Winter et al., 2007; Le et al., 2010;Dean et al., 2011). However, the proteins encoded by thesegenes do not contain known functional domains other thanputative signal peptides, so no function has been ascribed tothem to date. Members of the UPF0540 protein family arestrongly conserved, as they share 79% amino acid sequenceidentity and 81% similarity with one another (Fig. 4). Fur-thermore, the loci that encode these proteins are tightlyclustered on chromosome 1, suggesting that the gene familymay have expanded through tandem duplication events.Due to the abundance of the UPF0540 proteins in the seedcoat, these genes were named TESTA ABUNDANT1(TBA1; At1g62000/Q39168), TBA2 (At1g62060/O04573),and TBA3 (At1g62080/O04575). Interestingly, a pep-tide from a fourth member of the UPF0540 family,At1g62220/O04587, also was detected in adherentmucilage (Supplemental Data Sets S1 and S3). How-ever, At1g62220/O04587 was identified with only onepeptide with a score below the cutoff for statistical sig-nificance. Due to the strong similarities in amino acidsequences and expression patterns between At1g62220/O04587 and the TBA proteins, At1g62220/O04587 wasnamed TBA-LIKE (TBAL).TBAs are small proteins (;150 amino acids) with

many conserved Ser and Thr residues predicted byNetOGlyc to be O-glycosylated (Steentoft et al., 2013;Fig. 4). These characteristics suggest that TBAs andTBAL may function as structural proteins that interactwith various polysaccharides in seed coat mucilage.

TBA Proteins Are Synthesized in the Developing SeedCoat Epidermis and Secreted to the Apoplast

Public microarray data suggest that TBA and TBALare expressed uniquely in the seed coat, and reverse

transcription (RT)-PCR results are consistent with thispattern (Fig. 5). TBA and TBAL transcripts could onlybe detected in siliques (Fig. 5A) and, more specifically,in the 7- and 10-d post anthesis (DPA) seed coat (Fig.5B). TBA2 expression levels are by far the highest andpeaked at 7 DPA (coinciding with mucilage synthesis;Fig. 5B), whereas the expression levels of the remaininggenes were lower and peaked at 10 DPA (coincidingwith columella synthesis; Fig. 5B).

To verify the expression pattern of TBA and TBAL,reporter assays were performed on tissues of plantscarrying chimeric genes encoding the GUS gene underthe control of the TBA and TBAL native promoters.Consistent with RT-PCR data, GUS activity could bedetected in developing seeds at 7 and 10 DPA (Fig. 6B)but not in seedlings, leaves, stems (Fig. 6A), and em-bryos (Fig. 6B). GUS under the control of TBA2pappeared earlier compared with other promoters, al-though in general, all TBA and TBAL promoters wereactive by 10 DPA (Fig. 6B). These data support thehypothesis that all four promoters are active primarilyin the seed coat. The fact that the TBA promoter-GUSpatterns mirror the presence of TBA transcripts impliesthat the expression of the TBA genes is largely regulatedby their upstream cis-regulatory elements.

Several transcription factors are known to regulatethe differentiation of seed coat epidermis and the syn-thesis of seed coat mucilage. Some of these masterregulators include NAC-REGULATED SEED MOR-PHOLOGY1 (NARS1), NARS2, MUM1, MYELO-BLASTOSIS61 (MYB61) and TRANSPARENT TESTAGLABRA1 (TTG1; Koornneef, 1981; Penfield et al.,2001; Kunieda et al., 2008; Huang et al., 2011). Since theTBA genes are expressed exclusively in the seed coat,we asked whether they are under the control of thesetranscription factors. RT-PCR results showed that TBAand TBAL transcripts were absent in developing seedsof both the nars1 nars2 doublemutant and ttg1-1 (Fig. 7),which suggests that NARS1/NARS2 and TTG1 are allrequired for TBA and TBAL expression. Intriguingly,TBA2 transcripts appear to be absent in the Landsbergerecta ecotype, suggesting that there may be TBA ex-pression variation among different natural accessions.

The subcellular localization of TBAs was characterizedusing C-terminally tagged citrine-yellow fluorescent pro-tein (cYFP) TBA translational fusion constructs driven by

Figure 4. Amino acid sequences of the TBA pro-teins. Amino acid sequence alignment is shownfor TBA1, TBA2, and TBA3. Dark gray highlightsamino acid residues that are identical, and lightgray highlights amino acid residues that are simi-lar. Underlined residues denote signal peptidespredicted by SignalP (http://www.cbs.dtu.dk/services/SignalP/). Boldface S and T residues arepredicted by NetOGlyc (http://www.cbs.dtu.dk/services/NetOGlyc/) to be O-glycosylated.








http://www.cbs.dtu.dk/services/SignalP/

http://www.cbs.dtu.dk/services/SignalP/

http://www.cbs.dtu.dk/services/NetOGlyc/

http://www.cbs.dtu.dk/services/NetOGlyc/


their endogenous promoters. In agreement with theexpression data, cYFP signals were detected in the devel-oping seed coat. No cYFP signal was observed in 4-DPAseeds (Fig. 8). By 6DPA, TBA1-cYFP andTBA2-cYFP couldbe detected in the seed coat epidermal lateral cell walls andthe developing mucilage pockets. All three TBAs could bedetected in the mucilage pockets by 8 DPA (Fig. 8). By10 DPA, the cYFP signal was absent from the mucilagepocket but was observed in the developing columella (Fig.8). This expression pattern coincides spatiotemporally withthe TBA transcript and promoter activity patterns (Figs. 5and 6) and reinforces the characterization of TBA proteinsas mucilage proteins. Furthermore, cYFP fluorescence wasdetected only in the outer epidermal layer of the seed coat(Fig. 8), suggesting that TBA proteins may only be syn-thesized in mucilage-secretory cells.

Despite the fact that TBAs were initially identified inmature mucilage, TBA-cYFP fluorescence was absentfrom mucilage pockets by 10 DPA, which raised thepossibility that TBAsmight be unstable proteins. To testthis idea, immunoblot experiments were performed to

try to detect TBA-cYFP extracted from developing si-liques. Interestingly, full-length TBA-cYFP proteinswere difficult to detect in the siliques of TBA-cYFPtransgenic plants by immunoblotting. The abundanceof TBA-cYFP was quite variable among different lines,and TBA3-cYFP appears to be partially insoluble(Supplemental Fig. S5). These results suggest that TBA-cYFP proteins are likely unstable and may undergoproteolysis or other posttranslational modificationwithin the mucilage pocket.

TBA Proteins Are Likely Functionally Redundant

In an attempt to determine the function of the TBAproteins, we characterized tba loss-of-function mutants.Since the three TBA proteins are highly conserved andshare a similar expression pattern, we anticipated thatthey might be functionally redundant. Furthermore,because all these genes are closely linked on the chro-mosome, the construction of double, triple, and qua-druple tba mutants is relatively difficult. To overcomethese problems, an artificial microRNA (amiRNA)driven by the UBIQUITIN EXTENSION PROTEIN1promoter (UBQ1p) was designed to knock down allthree TBA homologs and TBAL simultaneously. WhenTBA1 transcript levels in developing siliques werequantified in 12 UBQ1p:TBA-amiRNA transgenic lines,four lines showed significant down-regulation of TBA1,but only line 5 showed significant down-regulation inall three TBA genes and TBAL (Fig. 9A). However, noneof the UBQ1p:TBA-amiRNA lines showed any mucilagedefects when their seeds were imbibed in water, 0.05 M

EDTA, 0.05 M CaCl2, or 0.5 M Na2CO3 followed by Ru-thenium Red staining (Fig. 9B). These results suggestthat either the TBA genes are functionally redundant,even at low levels of expression, or that the amiRNAknockdown lines possess mucilage phenotypes that arenot clearly discernible with Ruthenium Red staining.

DISCUSSION

Proteins Are an Integral Part of the Arabidopsis SeedCoat Mucilage

Arabidopsis seed coat mucilage is a specialized layerof the extracellular matrix composed of cell wall car-bohydrates arranged in a distinct structure in the apo-plast of seed coat epidermal cells. Because of itsaccessibility and dispensability, mucilage has beenused as a genetic model for studying the structure andfunction of the plant cell wall (Arsovski et al., 2009;Haughn and Western, 2012). Forward genetic analysishas enabled the identification of several proteins thatare secreted with mucilage and required for normalmucilage structure. To more comprehensively definethe array of proteins involved inmucilage structure andmodification, we used proteomic analysis to examinemucilage extruded by mature Arabidopsis seeds.The mucilage extracted by extensive shaking yielded

Figure 5. TBA and TBAL transcripts are found predominantly in thedeveloping seed coat. A, RT-PCR detection of TBA1, TBA2, TBA3, andTBAL transcripts in seedlings, roots, rosette and cauline leaves, stem,inflorescence, and siliques. CYTOSOLIC GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPC) transcripts are shown ascDNA loading controls. B, Quantitative RT-PCR results showing therelative expression levels of TBA1, TBA2, TBA3, and TBAL in emptysilique valves, 4-DPA seeds, 7-DPA seed coats, 7-DPA embryos,10-DPA seed coats, and 10-DPA embryos. Expression levels were rel-ative toGAPC transcript levels. n = 3, and error bars denote SD. A secondbiological replicate was processed with similar results.


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protein preparations that possessed a consistent arrayof secreted polypeptides relatively free of intracellularproteins. The 28 proteins identified in Col-0 seed coatmucilage by this approach may not be very numerous,but they represent the same classes of proteins found inthe cell wall proteomes of other tissue types, whichgenerally contain less than 100 proteins (Albenne et al.,2013). This suggests that the mucilage protein extrac-tion protocol is at least comparable with other cell wallproteome studies in terms of protein recovery rate.Furthermore, the mucilage proteins identified includeall proteins believed to be secreted to the mucilagepocket that are not membrane anchored: MUM2,SBT1.7, BXL1, and PER36 (Dean et al., 2007; Macquetet al., 2007b; Rautengarten et al., 2008; Arsovski et al.,2009; Kunieda et al., 2013). In addition, proteomicanalysis of mucilage extracts from the seeds of ap2mutants that fail to differentiate a seed coat epidermisshowed decreases among the most abundant mucilageproteins with the exception of PER36 (see below),which, unlike the others, localizes to the primary cellwall surrounding the mucilage. Therefore, we believethat our method is sufficiently robust in characterizingthe mature mucilage proteome.

Seed Coat Mucilage Is a Suitable Model for Cell WallProtein Analyses

Most of the proteins of mature seed coat mucilage arefunctionally similar to proteins found in primary cellwalls from other tissues (Albenne et al., 2013; Fig. 2),

consistent with the idea that mucilage and cell wallsshare many biosynthetic and functional processes. Seedcoat mucilage has the experimental advantage overother types of cell walls that it is actively extruded andcan be extracted without tissue homogenization andassociated cytoplasmic contamination (SupplementalData Sets S1–S3). All of the mucilage proteins identifiedcontain predicted signal peptides, while proteinswithout signal peptides detected in mucilage generallyscored poorly (Supplemental Data Set S3), suggestingthat this method indeed strongly favors apoplasticproteins. This reenforces mucilage as a strong model inwhich to study cell wall proteins, as it has markedly re-duced cytoplasmic contamination compared with othercell wall proteomes while retaining a comparable proteinrecovery rate (Albenne et al., 2013). However, because theextraction of mucilage requires its hydration-induced ex-trusion, the proteomic profile established for maturemucilage may not include proteins that are normallypresent only in early developmental stages. Characteriz-ing the mucilage proteins from developing seeds will re-quire other analytical strategies.

Seed Coat Mucilage Proteome Is a Specialized CellWall Proteome

Carbohydrate-active enzymes identified in the seedmucilage proteome include two enzymes previouslydetected by molecular genetic analyses. The mucilageproteinsMUM2and BXL1 are required for the removal ofarabinogalactan side chains of cell wall polysaccharides

Figure 6. TBA and TBAL promoters are active exclusively in the seed coat. A, TBA1p:GUS, TBA2p:GUS, TBA3p:GUS, TBALp:GUS, and Col-0 seedlings, rosette leaves, stems, and inflorescences stained for GUS activities. Bars = 500 mm. B, TBA1p:GUS,TBA2p:GUS, TBA3p:GUS, TBALp:GUS, and Col-0 siliques and developing seeds at 4, 7, and 10 DPA stained for GUS activities.The 10-DPA seed coats and embryos were dissected and stained separately, as shown in the two columns at right. Bars = 500 mmfor siliques and 100 mm for dissected seed coats and embryos.








and for proper mucilage extrusion (Dean et al., 2007;Macquet et al., 2007b; Arsovski et al., 2009). Four othercarbohydrate-active enzymes not previously known to bemucilage associated also were identified, includingHEXO3 (At1g65590/Q8L7S6). HEXO3 has been shownto be involved in the removal of GlcNAc residues fromglycoproteins and in the formation of paucimannosidicN-glycan (Gutternigg et al., 2007; Liebminger et al., 2011;this study). HEXO3 localizes primarily to the plasmamembrane, although aminor fraction has been suggestedto be soluble in the apoplast, which would be consistentwith our findings (Liebminger et al., 2011). However, theoverall biological role of HEXO3 and its orthologHEXO1remains unknown, as no defects in growth and stressresponses were found in their respective mutants(Liebminger et al., 2011).

Proteases are commonly found in cell walls, and fourwere detected in the mucilage proteome. SBT1.7 hasbeen identified previously by its mutant mucilagephenotype of defective extrusion and altered homo-galacturonan methylation state. It has been suggestedthat SBT1.7 participates in the removal of the inhibitordomain from a pectin methylesterase (Rautengartenet al., 2008). Two other proteases detected in mucilagehave been connected to roles in other tissues. Ectopicexpression of ASPG1 (At3g18490/Q9LS40) enhancesabscisic acid (ABA)-induced stomata closure, reactiveoxygen species production, and drought resistance (Yaoet al., 2012), and ASPG1 expression also is induced byABA (Yao et al., 2012). RD21a (At1g47128/P43297) isup-regulated during drought stress, suggesting that italso may be connected to ABA-regulated processes(Koizumi et al., 1993). In addition, RD21a is known tofacilitate apoptosis, which the outer seed coat epidermalcells eventually undergo at the end of seed development

(Lampl et al., 2013). However, no obvious mucilage de-fects were observed in either aspg1 or rd21a seeds.

Oxidoreductases can potentially modify cell wallcomponents either by regulating the production andturnover of reactive oxygen species or by participating inthe oxidative modification of other cellular metabolites.Mucilage proteins of this category include PER36, whichwas shownpreviously to facilitatemucilage extrusion byweakening the primary cell wall (Kunieda et al., 2013).PER36 localizes to the radial and tangential primarywalls of themucilage pockets but was not detected in themucilage itself (Kunieda et al., 2013). Therefore, it islikely that PER36 is not part of the mucilage proteomebut, rather, a contaminant from the primary wall. Con-sistent with this, PER36was the only protein found to bemore abundant in mum2-10 seed surface extracts com-pared with Col-0 (Fig. 3B). Primary cell wall proteinssuch as PER36 would be expected to be overrepresentedin the proteome extracted from mum2-10 seeds, sincemucilage extrudes very poorly from this mutant.

Proteins involved in lipid metabolism also are com-mon in cell wall proteomes (Albenne et al., 2013). Theseproteins are likely involved in the synthesis and mod-ification of cuticles deposited outside the cell wall. Inseed coat mucilage, proteins involved in lipid metabo-lism associate exclusively with the adherent layer,making it the only obvious distinction between theproteomes of the two mucilage layers (Fig. 2A). Sincethe seed coat epidermis likely has a cuticle (Watanabeet al., 2004; Panikashvili et al., 2009) and the primarywall remains attached to the top of the columella em-bedded in the adherent layer, these proteins may beinvolved in the synthesis/modification of a seed coatcuticle. The fact that proteins involved in lipid metab-olism are found only in the adherent layer suggests thatthey are very strongly bound to the primary cell wall,either covalently cross-linked with other cell wall pol-ymers or perhaps anchored by hydrophobic interac-tions with the cuticle. In contrast, PER36 was observedin the primary cell wall, but it was found in both ad-herent and nonadherent mucilage in our analysis,suggesting that it is less strongly bound to the cell wallthan the proteins involved in lipid metabolism.

One difference between themucilage and other cellwallproteomes is the apparent lack of structural proteins inmucilage. No extensins and Hyp-rich glycoproteins, typ-ically major components of cell wall proteomes, were ob-served (Jamet et al., 2008a; Albenne et al., 2013), althoughthe Leu-rich repeat family protein At3g24480/Q9LHF1might play a structural role. Furthermore, the TBAproteinstructure (short, no identifiable protein domains, potentialglycosylation sites) and abundance (see below) are char-acteristics consistent with those of structural proteins, al-though we have no direct evidence supporting thishypothesis. Arabinogalactan proteins also may func-tion as structural proteins, as has been suggested forARABINOXYLAN PECTIN ARABINOGALACTANPROTEIN1 (Tan et al., 2013). Two fasciclin-like ara-binogalactan proteins, FLA10 (At3g60900/Q9LZX4)and FLA17 (At5g06390/Q66GR0), were identified in

Figure 7. TBA and TBAL expression requires NARS1, NARS2, andTTG1. RT-PCR detection is shown for TBA and TBAL transcripts in7-DPA seeds of nars1 nars2, mum1-1, myb61-1, ttg1-1, and their re-spective ecotype backgrounds. GAPC transcripts are shown as cDNAloading controls. Ler, Landsberg erecta.


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mucilage, but their loss-of-function phenotypes did notprovide any insight into a possible structural role. Sur-prisingly, SALT OVERLY SENSITIVE5 (SOS5/FLA4;At2g46550), the only FLA known to be required fornormal mucilage structure (Harpaz-Saad et al., 2011;Griffiths et al., 2014), was not identified as a componentof thematuremucilage proteome. Itmay be possible thatSOS5 impacts mucilage structure indirectly, perhaps byfacilitating matrix polysaccharide biosynthesis in theGolgi or acting as a GPI-anchored carrier of carbohy-drates to the apoplast.Despite the identification of numerous newmucilage

proteins, in addition to the four proteins (MUM2, BXL1,PER36, and SBT1.7) previously known to regulatemucilage extrusion and structure (Dean et al., 2007;Macquet et al., 2007b; Rautengarten et al., 2008;Arsovski et al., 2009; Kunieda et al., 2013), the biological

roles of the new proteins in mucilage remain unknown.Analysis of loss-of-function mutants for many of thegenes encoding these proteins failed to identify muci-lage extrusion and morphology defects. This may re-flect functional redundancy. Alternatively, it mayindicate that these mucilage proteins play roles that donot directly impact the extrusion, adherence, or struc-ture of the mature mucilage and that, therefore, do notgenerate a loss-of-function phenotype readily detect-able by Ruthenium Red staining and light microscopy.

TBA Proteins Are Seed Coat-Specific Proteins withPotential Roles in Seed Coat Differentiation

Among the mucilage proteins most frequently iden-tified in this study are a family of three uncharacterized

Figure 8. TBA proteins are secreted tothe seed coat epidermis apoplast.Confocal microscopy images denotethe localization of cYFP-tagged TBA1,TBA2, and TBA3 in developing seedcoats driven by their respective endog-enous promoters. C, Columella; CC,cytoplasmic column; M, mucilagepockets. Bars = 10 mm.





proteins designated TBA, which are highly and specif-ically expressed in the seed coat epidermis during lateseed development (Figs. 5 and 6). The expression ofTBAs requires the seed coat differentiation master reg-ulators NARS1, NARS2, and TTG1 (Fig. 7) and coin-cides temporally and spatially with mucilage synthesisand secretion, while ectopically expressed TBAs wereshown to localize to the seed coat mucilage and thecolumella (Fig. 8). Since TBAs are abundant and arepredicted to be heavily glycosylated while lackingknown functional domains, they may function as

structural proteins and cross-link other cell wall poly-saccharides (Fig. 4). However, our attempts to useamiRNA to knock down all of the TBA homologs si-multaneously failed to produce a Ruthenium Red-stained mucilage mutant phenotype (Fig. 9), so thishypothesis remains to be validated. It is interesting that,following the completion of mucilage synthesis duringcolumella formation, cYFP fluorescence in the mucilagepocket abruptly disappears (Fig. 8; 10 DPA), leavingfluorescence only in the columella. These observationssuggest that the TBA-cYFP proteins may be actively

Figure 9. Down-regulation of TBA and TBAL doesnot affect mucilage extrusion. A, Quantitative RT-PCR analysis of the expression of TBA and TBAL in10-DPA siliques from four independent UBQ1p:TBA-amiRNA lines and Col-0. All expressionlevels were relative to GAPC and were then nor-malized to Col-0 expression levels. n = 3, and er-ror bars denote SD. Asterisks denote transcriptlevels significantly different from the wild type atP, 0.05 (*) and P, 0.01 (**). B, Ruthenium Red-stained seed coat mucilage from the four inde-pendent UBQ1p:TBA-amiRNA lines shown in A.Seeds were imbibed in water, 0.05 M EDTA, 0.5 M

Na2CO3, or 0.05 M CaCl2 prior to staining. Bars =100 mm.


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degraded, although we cannot rule out the possibilitythat the fluorescence of the intact protein is beingquenched due to changes in the chemical environmentof the mucilage pocket.The seed coat specificity and relative strength of the

TBA promoters make them valuable tools for seed coatmucilage studies. Since the seed coat mucilage is notessential to plant fitness under laboratory conditions, itcan tolerate genetic perturbation, which has made it apowerful genetic model for cell wall analysis. Thespecificity of the TBA promoters makes them suitabletools to genetically manipulate the seed coat epidermisin general and seed mucilage specifically. AnotherArabidopsis seed coat-specific promoter, from theDIRIGENT PROTEIN1 gene (DP1), differs from ex-pression of the TBA promoters in that DP1 is expressedin both the epidermal and palisade cell layers and peaksin expression during midseed development (Esfandiariet al., 2013). Since the TBA promoters are active only inthe epidermis late in seed development, the TBA andDP1 promoters complement each other by coveringdifferent temporal and spatial domains.In summary, a novel method was developed to ex-

tract and detect proteins integral to the Arabidopsisseed coat mucilage. A total of 28 proteins were identi-fied in mature seed coat mucilage, mostly with pre-dicted functions consistent with a cell wall proteome.The protein profiles are largely similar between theadherent and nonadherent mucilage, with the excep-tion of lipid metabolism proteins that occur exclusivelyin the adherent layer mucilage. Three homologous,previously undescribed proteins we named TBA werehighly abundant in seed coat mucilage. Although theirfunctions remain to be determined, their seed coatepidermis-specific promoters should prove to be usefultools for targeted gene expression.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Most Arabidopsis (Arabidopsis thaliana) plants used in this study were de-rived from the Col-0 ecotype, except for ttg1-1, which is derived from theLandsberg erecta ecotype. Seeds were germinated on plates with Arabidopsismedium (Haughn and Somerville, 1986) at 7% agar and transferred to soilSunshineMix 4 (SunGro). Plants were grown with continuous fluorescent illu-mination of 80 to 140 mEm22 s21 at 20°C to 22°C. T-DNA insertion lines used inthis study were obtained from the ABRC and are listed in Supplemental TableS1. T-DNA insertion lines were selected with a PCR-based assay using primerslisted in Supplemental Table S2.

Seed Coat Mucilage Extraction

Dry seeds (40 mg) were imbibed with 800 mL of double-distilled water in amicrocentrifuge tube. The seeds were gently shaken on a tabletop shaker for 1 hat 120 rpm. Supernatants that contain the nonadherent mucilage were collected.The seeds were washed once with 200 mL of double-distilled water, which waspooled with the supernatant to form the nonadherent mucilage fraction. Toobtain the adherent mucilage, 800 mL of double-distilled water was added tothe seeds after extracting the nonadherentmucilage, and the seedswere securedhorizontally to a tabletop vortex and shaken at top speed for 3 h. The super-natants containing the adherent mucilage were collected. The seeds werewashed once with 200 mL of double-distilled water, which was pooled with the

supernatant as the adherent layer fraction. The mucilage samples were freezedried overnight and then chemically deglycosylated as described by Edge et al.(1981). Briefly, 15 mL of anisole (Sigma-Aldrich) and 135 mL of tri-fluoromethanesulfonic acid (Sigma-Aldrich) were added to one freeze-driedmucilage sample in a Reacti-vial. The samples were sealed and incubated at4°C for 2 h. Four microliters of 0.2% Bromophenol Blue was added to eachsample, and 60% pyridine (Sigma-Aldrich) was added drop wise to each mu-cilage sample on ice until the solution turned light blue. The neutralized mu-cilage samples were dialyzed overnight in double-distilled water with dialysistubing pore sizes of 3,500 to 5,000 D and then freeze dried overnight.

MS

Mucilage protein samples were resuspended in SDS-PAGE sample buffer andseparated briefly on a 10% SDS-PAGE gel until all of theMr markers just enteredthe resolving gel. The proteins were stained with blue silver (Candiano et al.,2004), and the entire lanewas excised from the gel as one gel slice. Protein sampleswere analyzed by tandem mass spectrometry (MS/MS) at the Centre for High-Throughput Biology Proteomics Core Facility at the University of British Co-lumbia. In brief, samples were subjected to reduction/alkylation with DTT/iodoacetamide followed by digestion with trypsin essentially as described byShevchenko et al. (1996). The resulting peptides were desalted and concentratedwith STAGE tips (Rappsilber et al., 2003) and analyzed by liquid chromatogra-phy-MS/MS on a linear-trapping quadrupole-Orbitrap mass spectrometer (LTQOrbitrap Velos) online coupled to an Agilent 1290 series HPLC device using ananospray ionization source (ThermoFisher Scientific) including a 2-cm-long100-mm i.d. fused silica trap column, a 20-cm-long 50-mm i.d. fused silica frittedanalytical column, and a 20-mm i.d. fused silica gold-coated spray tip (6-mm-diameter opening, pulled on a P-2000 laser puller from Sutter Instruments,coated on a Leica EM SCD005 Super Cool Sputtering Device). The trap column ispackedwith 5-mm-diameter Aqua C-18 beads (Phenomenex; www.phenomenex.com), while the analytical column is packed with 1.9 mm-diameter Reprosil-PurC-18-AQ beads (Dr. Maisch; www.dr-maisch.com). Standard 90-min gradientswere run from 10% to 32% buffer B (0.5% acetic acid and 80% acetonitrile) over51 min, then from 32% to 40% in the next 5 min, then increased to 100% over a2-min period, held at 100% for 2.5 min, and then dropped to 0% for another20min. TheHPLC system included anAgilent 1290 series pump and autosamplerwith thermostat. The thermostat temperature was set at 6°C. The sample wasloaded on the trap column at 5 mL min21, and the analysis was performed at0.1 mL min21. The LTQ-Orbitrap was set to acquire a full-range scan at 60,000resolution from350 to 1,600 Th in theOrbitrap to simultaneously fragment the topten peptide ions by CID and the top five peptide ions by HCD (resolution, 7,500)in each cycle in the LTQ (minimum intensity, 1,000 counts). Parent ionswere thenexcluded fromMS/MS for the next 30 s. Singly charged ionswere excluded, sincein electrospray ionization (ESI) mode, peptides usually carry multiple charges.The Orbitrap was continuously recalibrated using lock-mass function. For massaccuracy, the error of mass measurement is typically within 5 ppm and is notallowed to exceed 10 ppm.

For quantitative analyses, Col-0 mucilage along with mum2-10 and ap2-7seed surface extracts were prepared from 80 mg of seeds using the protocoldescribed above for nonadherent mucilage. All protein samples were reduced/alkylated and digested as described above and then dimethylated with light,medium, and heavy formaldehyde. Col-0 samples were labeled with lightformaldehyde for all replicates. mum2-10 samples were labeled with mediumformaldehyde for replicates 1 and 2 and with heavy formaldehyde for replicate3. ap2-7 samples were labeled with heavy formaldehyde for replicates 1 and2 and with medium formaldehyde for replicate 3. Samples from all three gen-otypes were pooled before theMS analysis. For replicates 1 and 2, samples wereanalyzed using the LTQ Orbitrap Velo as described above. For replicate 3,samples were analyzed using the Impact II quadrupole-time of flight massspectrometer (Bruker Daltonics) online coupled to an Easy nano LC 1000 HPLCdevice (ThermoFisher Scientific) using a captive spray nanospray ionizationsource (Bruker Daltonics) including a 2-cm-long 100-mm i.d. fused silica frittedtrap column and a 75-mm i.d. fused silica analytical column with an integratedspray tip (6–8-mm-diameter opening pulled on a P-2000 laser puller from SutterInstruments). The trap column is packed with 5-mm Aqua C-18 beads(Phenomenex; www.phenomenex.com), while the analytical column is packedwith 1.9-mm-diameter Reprosil-Pur C-18-AQ beads (Dr. Maisch; www.dr-maisch.com). The analytical column was held at 50°C by an in-house con-structed column heater. Samples were resuspended and loaded in buffer A(0.1% aqueous formic acid). Standard 45-min gradients were run from 10% to60%buffer B (0.1% formic acid and 80% acetonitrile) over 28min, then increasedto 100% over 2 min, and held at 100% for 15 min. The liquid chromatograph







http://www.phenomenex.com


http://www.dr-maisch.com





thermostat temperature was set at 7°C. The sample was loaded on the trapcolumn at 850 Bar, and the analysis was performed at a flow rate of 0.25 mLmin21. The Impact II quadrupole-time of flight mass spectrometer was set toacquire in a data-dependent auto-MS/MS mode with inactive focus frag-menting the 20 most abundant ions (one at the time at a rate of 18 Hz) after eachfull-range scan fromm/z 200 to 2,000 Th (at a rate of 5Hz). The isolationwindowforMS/MSwas 2 to 3 Th depending on parent ionmass-to-charge ratio, and thecollision energy ranged from 23 to 65 eV depending on ion mass and charge.Parent ions were then excluded from MS/MS for the next 0.4 min and recon-sidered if their intensity increased more than 5 times. Singly charged ions wereexcluded, since in ESI mode, peptides usually carry multiple charges. Strictactive exclusion was applied. For mass accuracy, the error of mass measure-ment is typically within 5 ppm and is not allowed to exceed 10 ppm. The nanoESI source was operated at 1,700 V capillary voltage, 0.20 Bar nano busterpressure, 3 L min21 drying gas, and 150°C drying temperature.

MS Data Analyses

For all qualitative analyses and replicates 1 and 2of the quantitative analyses,liquid chromatography-MS/MS data were processed with Proteome Discov-erer version 1.2 (ThermoFisher Scientific) and then searched against theUniprot-Swissprot Arabidopsis database using theMASCOT algorithmversion2.4 (Perkins et al., 1999; http://www.matrixscience.com). The database con-tains 12,069 sequences; no contaminants were added in the search space. Thefollowing parameters were applied: peptide mass accuracy, 10 ppm; fragmentmass accuracy, 0.6 D; trypsin enzyme specificity, fixed modifications, carba-midomethyl, variable modifications, Met oxidation; deamidated N, Q, andN-acetyl peptides, ESI-TRAP fragment characteristics. Only those peptides withIonScores exceeding the individually calculated 99% confidence limit (as op-posed to the average limit for the whole experiment) were considered as ac-curately identified. Proteome Discoverer parameters were as follows: eventdetector, mass precision of 4 ppm (corresponds to extracted ion chromatogramsat 612 ppm max error); S/N threshold, 1; quantitation method, ratio calcula-tion; replace missing quantitation values with minimum intensity, yes; usesingle peak quantitation channels, yes; protein quantification, use all peptides,yes. In order for a protein to be considered a truemucilage protein in qualitativeanalysis, it must be identified in at least two out of the three biological replicateswith MASCOT protein scores .40 (a score of $25 corresponds to a false dis-covery rate of #5%), and identified in at least one out of the three biologicalreplicates with two or more unique peptides.

For replicate 3of thequantitativeanalysis, dataanalysiswasperformedusingMaxQuant 1.5.3.30 (Cox and Mann, 2008) with the Arabidopsis protein se-quence database plus common contaminants. The search was performed usingthe following parameters: peptide mass accuracy, 10 ppm; fragment mass ac-curacy, 0.05 D; trypsin enzyme specificity, fixed modifications, carbamido-methyl, variable modifications, Met oxidation; and N-acetyl proteins. Onlythose peptides exceeding the individually calculated 99% confidence limit (asopposed to the average limit for the whole experiment) were considered asaccurately identified. Relative protein levels from mum2-10 and ap2-7 seedsurface extracts were normalized to Col-0 nonadherent mucilage. Proteins thatcould be detected and quantified in all three replicates were analyzed. A two-tailed Student’s t test was used to determine the statistical significance of rel-ative protein level differences between mum2-10, ap2-7, and Col-0.

Expression Analyses

RNA was extracted from various plant tissues using Trizol reagent (LifeTechnologies), except that siliqueswereprocessedwith theRNAqueousTotalRNAIsolation Kit (Ambion) while developing seed coats and embryos were processedwith the RNAqueous-Micro Total RNA Isolation Kit (Ambion) according to themanufacturer’s instructions. cDNA synthesis was carried out using SuperScript IIreverse transcriptase (Life Technologies) according to the manufacturer’s instruc-tions. Quantitative PCR (qPCR) was performed using iQ SYBR Green Supermix(Bio-Rad) and the primers listed in Supplemental Table S3. The qPCRs wereassayed with the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad).GAPC transcripts were used as an internal control. A two-tailed Student’s t testwas used to determine the statistical significance of differences in TBA and TBALexpression levels between the amiRNA lines and the wild type.

For gene expression analysis of TBAs in transcription factor mutants, RNAextraction and cDNA synthesis were performed as described previously(Kunieda et al., 2013). PCR was performed using Mango-Taq polymerase(Bioline) and the same primers that were used for qPCR.

Generation of Transgenic Plants

The TBAp:GUS constructs were generated using the pBI101 vector, with thepromoter fragments amplified from Col-0 genomic DNA. The TBAp:TBA-cYFPtranslational fusion constructswere assembled in the citrine-pCambia1300 vectoras described by Debono et al. (2009), with the promoter and coding regionfragments amplified from Col-0 genomic DNA. The TBA amiRNA constructswere designed and built as described by Schwab et al. (2006) using the UBQ1-pCambia1300 vector as described by Ambrose et al. (2011). Primers used forthese constructs are listed in Supplemental Table S3. The TBA and TBAL pro-moters were defined as the DNA sequence extending upstream of the TBA startcodon to the next annotated gene, not including pseudogenes, to capture asmuch of the promoter region as possible without introducing another gene.TBA1p is ;0.5 kb, TBA2p is ;1.3 kb, TBA3p is ;1.8 kb, and TBALp is ;1.4 kblong. Col-0 Arabidopsis plants were transformed using the floral dip method(Clough and Bent, 1998); at least 20 independent transgenic lines were selectedfor each construct. Results from at least three representative lines are shown.

Microscopy

For seed coat mucilage staining with Ruthenium Red, ;20 dry seeds wereimbibed in 1 mL of distilled, deionized water, 0.05 M EDTA, 0.05 M CaCl2, or 0.5MNa2CO3 for 1 h andwashed twice with double-distilledwater. The seeds werethen stained with 0.01% (w/v) Ruthenium Red (Sigma-Aldrich) for 1 h andwashed once with double-distilled water. Seeds were imaged with a DFC450 Ccamera (Leica) on an Axioskop 2 upright light microscope (Carl Zeiss).

Histochemical GUS assays were performed essentially as described byEsfandiari et al. (2013). Tissue samples were vacuum infiltrated with GUSstaining solution (0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocya-nide, 20 mM Na2EDTA, and 0.1% [v/v] Triton X-100, supplemented with 1 mgmL21 5-bromo-4-chloro-3-indolyl-b-D-glucuronide [Gold BioTechnology] in100 mM phosphate buffer [pH 7]), incubated at 37°C for 16 h, and then washedseveral times with 75% ethanol. Tissues were imaged with a DP72 camera(Olympus) mounted on an SZX10 stereomicroscope (Olympus).

All confocal images were acquired from an Ultraview VoX Spinning DiskConfocal System (PerkinElmer).

For cellulose staining, seeds aftermucilage extractionwere stainedwith 0.1%(w/v) Calcofluor White for 5 min and then washed twice with double-distilledwater.The seedswere inspectedunderUVlightwith theAxioskop2microscope.

Immunoblot Analyses

Developing siliques were ground in liquid nitrogen and added to extractionbuffer containing 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1 mM EDTA, 10% (v/v)glycerol, 1% Triton X-100, 1 mM PMSF, and protease inhibitor cocktail (Roche).The homogenates were centrifuged at 15,000 rpm for 10 min at 4°C, and the su-pernatant was collected. Protein concentrations were determined by Bradfordassay (Bio-Rad), and 100-mg protein sampleswere electrophoretically resolved by12% SDS-PAGE. cYFP fusion proteins were detected using a mouse anti-GFPpolyclonal antibody (Roche) and horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody (Santa Cruz Biotechnology). ECL Prime western-blotting detection reagent (GE Healthcare) was used for target detection.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under the following accession numbers: At1g65590/Q8L7S6, HEXO3;At5g63800/Q9FFN4, MUM2; At5g49360/Q9FGY1, BXL1; At3g50990/Q9SD46, PER36; At5g67360/O65351, SBT1.7; At3g18080/Q9LV33, BGLU44;At1g65590/Q8L7S6, HEXO3; At3g18490/Q9LS40, ASPG1; At3g07390/Q94BT2, AIR12; At1g47128/P43297, RD21a; At5g08260/Q9LEY1, SCPL35;At1g62000/Q39168, TBA1; At1g62060/O04573, TBA2; At1g62080/O04575,TBA3; At5g63140/Q9FMK9, PAP29; At5g06390/Q66GR0, FLA17; At3g60900/Q9LZX4, FLA10; At2g28470/Q9SCV4, BGAL8, At4g23560/Q9SUS0, GH9B15;At5g59310/Q9LLR6, LTP4; At3g08770/Q9LDB4, LTP6; At3g04170/Q9M8X3,RmlC-like cupin superfamily protein; At5g45670/Q9FK75, GDSL motif ester-ase/acyltransferase/lipase; At3g16370/Q9LU14, GDSL motif esterase/acyl-transferase/lipase; At1g75900/Q94CH6, GDSL motif esterase/acyltransferase/lipase; At4g26790/Q8VY93, GDSL motif esterase/acyltransferase/lipase;At4g28780/Q9SVU5, GDSL motif esterase/acyltransferase/lipase; At3g24480/Q9LHF1, Leu-rich repeat family protein.


Tsai et al.


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Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1.Mucilage extraction removes pectin and cellulosefrom the seed coat.

Supplemental Figure S2. Expression (RT-PCR) of genes mutated byT-DNA insertion.

Supplemental Figure S3. Imbibed seeds from plants homozygous for aT-DNA insertion in genes encoding proteins found in seed mucilage.

Supplemental Figure S4. TBA and TBAL expression patterns in variousplant tissues.

Supplemental Figure S5. Detection TBA-cYFP by immunoblotting.

Supplemental Table S1. T-DNA insertion lines used in this study.

Supplemental Table S2. Primers used for mucilage protein T-DNA lineanalyses in this study.

Supplemental Table S3. Primers used for TBA and TBAL constructs in thisstudy.

Supplemental Data Set S1. Col-0 adherent mucilage protein MS data.

Supplemental Data Set S2. Col-0 nonadherent mucilage protein MS data.

Supplemental Data Set S3. Summary of Col-0 mucilage protein identifi-cation.

Supplemental Data Set S4. mum2-10 and ap2-7 mucilage protein quantifi-cation MS data relative to Col-0.

Supplemental Data Set S5. Summary of mum2-10 and ap2-7 mucilage pro-tein quantification relative to Col-0.

ACKNOWLEDGMENTS

We thank Suzanne Perry, Jamie Hackworth, and JennyHyung-MeeMoon ofthe University of British Columbia Centre for High-Throughput BiologyProteomics Core Facility for technical assistance, data analyses, and equipmentuse; the University of British Columbia Bioimaging Facility for technicalassistance and equipment use; and Ikuko Hara-Nishimura and Dr. TomooShimada (Department of Botany, Kyoto University) for providing the nars1nars2 mutant.

Received October 20, 2016; accepted December 15, 2016; published December21, 2016.

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