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# Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm

M. Suliman, Anne-Laure Chateigner-Boutin, M. Francin-Allami, A. Partier, B. Bouchet, J Salse, C. Pont, J. Marion, H. Rogniaux, D. Tessier, et al.

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► To cite this version:

M. Suliman, Anne-Laure Chateigner-Boutin, M. Francin-Allami, A. Partier, B. Bouchet, et al. Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm. Journal of Proteomics, 2013, 78, pp.508-521. 10.1016/j.jprot.2012.10.021. hal-03261448

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Identification of Golgi-localized glycosyltransferases involved in cell wall synthesis of wheat endosperm

M. Sulimana, AL. Chateigner-Boutinª, M. Francin-Allamiª, A. Partierᵇ, B. Bouchetª, J. Salseᵇ, C. Pontᵇ, J. Marionᶜ, H. Rogniauxª, D. Tessierª, F. Guillonª, C. Larréª*

ªINRA, UR1268 Biopolymères, Interactions Assemblages, F-44316 Nantes, France.

ᵇINRA, UMR1095 Génétique, Diversité & Ecophysiologie des Céréales, F-63100 Clermont-Ferrand, France.

ᶜInstitut des Sciences du Végétal, F-91198 Gif-sur-Yvette, France.

MS and ALCB contributed equally to the work.

Abstract:

Plant cell walls are complex structures critical for plant fitness and valuable for human nutrition as dietary fibre and for industrial uses such as biofuel production. The cell wall polysaccharides in wheat endosperm consist of two major polymers, arabinoxylans and beta-glucans, as well as other minor components. Most of these polysaccharides are synthesized in the Golgi apparatus but the mechanisms underlying their synthesis have yet to be fully elucidated and only a few of the enzymes involved have been characterized. To identify actors involved in the wheat endosperm cell wall formation, we used a subcellular fractionation strategy to isolate Golgi-enriched fractions from endosperm harvested during active cell wall deposition. The proteins extracted from these Golgi-enriched fractions were analysed by LC-MS/MS. We report the identification of 1135 proteins among which 64 glycosyltransferases distributed in 17 families. Their potential function in cell wall synthesis is discussed. In addition, we identified 63 glycosylhydrolases, some of which may be involved in cell wall remodeling. Several glycosyltransferases were validated by showing that when expressed as fusion proteins with a fluorescent reporter, they indeed accumulate in the Golgi apparatus. Our results provide new candidates potentially involved in cell wall biogenesis in wheat endosperm.

Keywords:
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Glycosyltransferases / Golgi apparatus / subcellular fraction / wheat endosperm / proteomics

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Abbreviations:

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AX arabinoxylan, MLG (1-3) (1-4) β-D-glucan or mixed-linked glucan, GT glycosyltransferases, GH glycosylhydrolase

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# 1 Introduction

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Plant cells are surrounded by cell walls composed of polysaccharides, proteins and, in some specialized cells, various non-carbohydrate substances (e.g., lignins). Cell wall composition varies between species and between cell types and developmental stages within a given plant species. Cell wall polysaccharides have been grouped into cellulose, hemicelluloses and pectins [1]. Pectins and hemicelluloses are components of the wall 'matrix', within which the cellulose framework is embedded. The walls of most dicots and non-grass monocots (type I walls) contain about equal amounts of xyloglucan and cellulose. Grasses are noteworthy for the unusual composition of their cell walls (type II walls), which contain less pectin and xyloglucan, but more heteroxylan than the walls of other higher plants. The cell wall can accumulate extensive interconnecting networks of phenylpropanoids, particularly as the cells stop expanding and start developing secondary cell walls. The chemical structure of the components and the way that they are assembled are key factors underlying cell wall properties. These properties are important not only for plant fitness but also for the processing quality of crop materials (e.g., degradation potential for biofuel production and bread-making quality of wheat flour).

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The cell wall composition of wheat grain endosperm is very specific, essentially composed of arabinoxylan (AX) (70%) and (1-3) (1-4) β-D-glucan or mixed-linked glucan (MLG) (20%) with minor amounts of cellulose (4%) and glucomannan (7%) [2, 3]. The synthesis of cell wall polysaccharides is activated during wheat grain development. Drastic changes in cell wall composition and structure were observed in the developing endosperm [4-6]. In particular, a transient accumulation of (1-3) β-D-glucan (callose) and xyloglucan has been reported [4, 7].

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The metabolism of cell wall polysaccharides requires the intervention of numerous enzymes. Enzymes acting on carbohydrates are listed and classified on the basis of their sequence similarities and activities in the Carbohydrate-Active enZymes
database (CAZy, http://www.cazy.org/) [8]. Currently, CAZy contains 94
glycosyltransferase (GT) families covering 122 enzymatic activities, 130
glycosylhydrolase (GH) families covering 158 activities, as well as 22 families of
polysaccharide lyases, 16 of carbohydrate esterases (CEs) and 64 families of
carbohydrate-binding modules (CBMs). Plants are under-represented in this database,
essentially because there are less fully-sequenced genomes of plant species
compared to other kingdoms such as bacteria. However, in the sequenced model
*Arabidopsis thaliana*, about 3.3% of all of the genes encode carbohydrate-active
enzymes [9, 10].

Cell wall polysaccharides are synthesized from activated precursors (nucleotide
sugars) by enzymatic complexes. Several groups have accumulated evidence for two
major subcellular locations for cell wall polysaccharide synthesis. Cellulose microfibrils
and callose are synthesized at the plasma membrane surface and the polysaccharides
are deposited directly into the cell wall [11-13]. Pectin and hemicellulose are
synthesized in the Golgi apparatus to then be transported to the wall [14-18].

The GT superfamily contains enzymes that synthesize polysaccharides such as starch
and cell wall polysaccharides. GTs are also involved in the glycosylation of many other
substrates such as proteins, lipids and secondary metabolites. Several GTs involved
in cell wall polysaccharide synthesis have been localized in the Golgi apparatus [19-
21]. GTs involved in protein glycosylation may also be localized in the Golgi apparatus
and in the endoplasmic reticulum (ER) [22, 23], whereas GTs involved in starch
synthesis were localized in plastids [24].

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To gain access to the cellular machinery involved in the synthesis of the cell wall
hemicelluloses in the wheat grain endosperm, we used a subcellular proteomic
approach that targeted the Golgi apparatus to focus on the direct actors of the
polysaccharide biosynthesis, i.e., the enzymes. Subcellular fractionation has been
widely described across all cell types and tissues, and has been shown to be
particularly efficient for organelles such as mitochondria [25] and chloroplasts [26],
leading to pure fractions of these organelles. However, subcellular proteomics remains
challenging for the endomembrane system. In the case of the Golgi apparatus and the
ER, their similar density makes it very tedious to isolate them as pure fractions.
Moreover, the large number of proteins trafficking from the ER to the Golgi apparatus
inevitably leads to the identification of ER proteins, even in an ER-free Golgi
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preparation [27]. Efficiently monitoring the subcellular fractionation and assessing the
possible contamination of the isolated fractions are additional difficulties [23, 28].

Finally, many GTs are membrane proteins characterized by issues such as poor
solubility, low abundance and recalcitrance to classical proteomic techniques.

In the present work, we have developed a subcellular strategy based on wheat
endosperm Golgi fractionation from developing grain harvested at a stage where cell
wall polysaccharides are accumulating. We partitioned integral membrane and water
soluble proteins associated with the endomembranes. Using mass spectrometry, we
identified 1135 proteins, 64 of which were predicted to be GTs involved in glycosylation
processes in the wheat endosperm. We also identified 63 GHs potentially involved in
the remodelling or degradation of the endosperm carbohydrates. The proteins
identified comprise Golgi residents and proteins in transit in the Golgi apparatus.

## 2. Materials and methods

### 2.1. Plant materials:

The plants *Triticum aestivum* cv. Recital were grown in pots in a greenhouse under conditions of natural day length (UMR Amélioration des Plantes et Biotechnologies Végétales, INRA-Rennes, France). To harvest grains at defined developmental stages, individual ears were tagged at flowering. Seed development was calculated on the basis of cumulated temperature in Celsius degrees days (°D) after flowering. Grains were harvested between 250 and 275°D and endosperms were isolated manually and maintained in buffer A (250 mM sucrose, 10 mM HEPES-NaOH, pH 7.4, 1 mM EDTA, 1 mM DTT and anti-protease cocktail) (Roche) on ice before further treatment.

For microscopy studies, the plants were grown in pots in a growth chamber at 23°C day/15°C night at 65% relative humidity under a 16h/8h day/dark photoperiod (UPR 2355/IFR 87, Gif Sur Yvette, France). Wheat grains were harvested at 250°D.

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### 2.2. Electron transmission microscopy

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#### 2.2.1 Sample preparation

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Pieces of tissue (1 mm³) were sampled from halved wheat grains. Samples were prepared as follows for morphological assay. Samples were loaded into copper hats filled with 1-hexadecene and high-pressure frozen using a HPF-EM PACT 2 freezer (Leica, http://www.leica-microsystems.com). The hats were immediately stored in
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liquid nitrogen until the freeze substitution procedure was initiated. Samples were transferred to a freeze substitution automate (AFS2/FSP, Leica) and pre-cooled to -90°C. Samples were substituted in anhydrous acetone with 2% osmium tetroxyde at -90°C for 125 h. The temperature was gradually raised to -30°C for 60 h and stabilized for 12 h, then gradually raised to -4°C (34 h) and stabilized for 3 h. Samples were rinsed in anhydrous acetone several times and slowly infiltrated and embedded in Epon's resin.

For immunolabeling, samples were prepared as described by Chevalier et al. [29]. High-pressure freezing was performed with the HPF-EM PACT I freezer. Samples were then transferred to an AFS. Samples were finally embedded in London Resin White (LRW, LRWhite R1280 HARD GRADE, London Resin Company Limited) and polymerization was performed in the AFS apparatus at -15°C under UV light for 48 h.

## 2.2.2 Polysaccharide staining and immunolabeling

Periodic acid-thiosemicarbazide-silver proteinate staining (PATAg) [30] for polysaccharide detection.

Ultra-thin sections (80 nm) were cut from Epon's embedded samples using an ultramicrotome (MICROM MT-7000) equipped with a diamond knife and directly floated in 1% periodic acid (MERCK) solution for 30 min. They were then rinsed 5 times in deionized water before incubation in thiosemicarbazide (0.2%) (MERCK) diluted in 20% acetic acid for 17 h. Sections were rinsed 5 times in decreasing acetic acid concentrations (20, 10, 5 and 2.5%), 4 times in deionized water, and stained with 1% aqueous silver proteinate (PROLABO) in the dark for 30 min. After rinsing 5 times in deionized water, sections were mounted on copper grids. Sections were examined with a JEOL 1230 transmission electron microscope with an accelerating voltage of 80 KeV (IBISA/BioGenOuest Biopolymers, Interations, Structural Biology platform (BIBS), UR 1268 BIA, INRA Angers-Nantes).

### Immunolabeling

Ultra-thin sections (80 nm) were cut from LRW embedded samples and collected on nickel grids. Sections were incubated in a blocking solution of 3% (w/v) BSA in 20 mM PBS, pH 7.2, to block non-specific labeling for 30 min at room temperature. Sections were then incubated in a solution containing the mouse monoclonal antibody anti-AX1 [31] and the rabbit polyclonal antibody raised against the pea reversibly glycosylated polypeptide 1 (anti-RGP1) (a gift from K. Dhugga), diluted 1:20 and 1:2500,
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respectively, in 20 mM PBS supplemented with 1% BSA and 0.05% Tween-20 (Sigma-

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Aldrich) for 1 h at room temperature. The sections were extensively washed in buffer

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used for diluting the primary antibodies and then incubated 1 h at room temperature in

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the dark with goat-anti-mouse IgG or goat-anti rabbit conjugated with 1 nM colloidal

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gold complexes diluted 1:20 (v/v) in the respective buffer chosen for the primary

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antibodies. Labeling was then intensified with the silver enhancement kit (Aurion)

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according to the manufacturer's instructions. After washing, the grids were stained with

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2% uranyl acetate. Sections were examined with a JEOL 1230 transmission electron

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microscope with an accelerating voltage of 80 KeV.

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## 2.3. Preparation of the microsomal fraction

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Six experiments were conducted. For each experiment, approximately 20 g of

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endosperm were ground with a mortar and pestle on ice in buffer A and then

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homogenized on ice using a polytron (Kinematica AG, Dispersing and Mixing

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Technology) for two pulses of 7 s at 7500 rpm. The endosperm lysate was then

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centrifuged twice for 5 min at 2,200 g to separate cell debris. The supernatant was

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subsequently loaded on the top of an SW41 centrifuge tube that contained 8 mL of a

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18% iodixanol prepared from Optiprep solution (Sigma-Aldrich) and centrifuged at

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100,000 g for 2 h at 4°C (Beckman Coulter SW41 rotor). The interfacial fraction was

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collected as the microsome fraction.

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## 2.4. Generation of Golgi-enriched fractions

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Four mL of the recovered microsomal fraction were then collected from the interface

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and suspended in a final solution of 2.8 mM HEPES, pH 7.4, 0.28 mM EDTA, 0.28 mM

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DTT, 14% iodixanol and gently mixed to avoid organelle damage. These organelles

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were further sub-fractionated by generating an iodixanol gradient by ultracentrifuging

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at 200,000 g for 12 h in a Ti 70 rotor (Beckman Coulter). Fifteen fractions of 1.8 mL

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were harvested from the top of the gradient and their densities were determined using

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a refractometer (Maselli Misure; LR-01 Digital Refractometer). The protein

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concentration of each fraction was then determined using the NI™ Protein Assay (G-

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Biosciences).

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## 2.5. Separation of organelle membranes
The collected fractions, microsomal and Golgi-enriched fractions, included organelles and their associated proteins. Cold deionized water was added to each fraction to reach a final volume of 22 mL in a centrifuge bottle and ultracentrifuged at 100,000 g for 2 h. The pellet consisted of organelle membranes from which membrane proteins were extracted for further identification; they will be referred to as MP (membrane proteins), MPG when issued from Golgi-enriched fractions. The supernatants of the microsomal and fraction samples were extensively dialyzed against cold water at 4°C using a semi-permeable membrane with a molecular weight cut-off of 3,500 (Spectra Pro). The dialysis water was replaced several times over the course of three days at 4°C. The dialyzed proteins were then collected and lyophilized; these fractions will be referred to as AP (proteins associated with the membranes).

## 2.6. Monitoring of ER and Golgi enrichment in fractions by Western blot analysis

The hydrophobic nature of membrane proteins led us to choose an anionic detergent for their solubilization. Both membrane pellets and lyophilized AP were therefore solubilized in 100 µL of Tris-HCl buffer, pH 6.8, containing 2% (w/v) SDS, 10% (v/v) glycerol and bromophenol blue. Solubilized proteins were reduced by the addition of 5 µL of β-mercaptoethanol and separated by SDS-PAGE on 12% acrylamide gels. After separation, gels were electroblotted onto nitrocellulose membranes (Amersham Biosciences) in 25 mM Tris, 192 mM glycine, 0.1% SDS and 20% ethanol. Semi-dry transfer was achieved at 100 V for 1.5 h.

Membranes were washed in PBS solution, blocked at room temperature in 5% dry milk in PBS, 0.1% Tween-20, and then washed twice for 10 min in PBS, pH 7.4, 0.1% Tween-20. Two primary antibodies were used for incubation. The polyclonal antibody anti-RGP1 (see above for immunolabeling), which recognizes a Golgi-localized protein, was used at 1:10,000 dilution for 2 h at room temperature. The antibody anti-BiP2 (Binding immunoglobulin protein2, Agrisera AB, Sweden), specific for Arabidopsis ER, was used at 1:2,000 dilution for 2 h at room temperature. After washing 2 times in PBS, 0.1% Tween-20, the secondary antibodies, consisting of peroxidase-conjugated anti-rabbit IgG, were used at 1:5,000 and 1:200,000 dilution, respectively, for 2 h at room temperature. After washing, nitrocellulose membranes were incubated in a chemiluminescent substrate (Super Signal West Dura Extended Duration Substrate, Pierce) for 6 min and dried between two paper sheets according
1 to the manufacturer's instructions. Luminescence was then detected using a camera
2 (Luminescent Image Analyzer LAS 3000; Fuji Film).

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4 2.8. Analysis of peptides by mass spectrometry

5 Proteins extracted from fractions that exhibited a high Golgi apparatus content as
6 revealed by RGP1 were separated by SDS-PAGE on 12% acrylamide gels. The
7 migration was performed on 2 cm of gel in order to increase the efficiency of tryptic
8 digestion. After separation, proteins were stained with Coomassie Brilliant Blue G250
9 (Sigma-Aldrich), as described by Devouge et al. [32].

10 2.8.1. Protein digestion

11 For each SDS-PAGE lane, 20 bands were manually excised from the gels to be
12 hydrolyzed. In brief, the gel pieces were first washed three times in 50% (v/v) ACN/25
13 mM ammonium bicarbonate, and the proteins were then reduced using DTT and
14 alkylated with iodoacetamide prior to tryptic digestion, according to Larré et al. [33].

15 2.8.2. Liquid chromatography and mass spectrometry

16 Nanoscale capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS)
17 analyses of the digested proteins were performed using an Ultimate 3000 RSLC
18 system (Dionex) coupled with an LTQ-Orbitrap VELOS mass spectrometer (Thermo
19 Fisher). Chromatographic separation was conducted on a reverse-phase capillary
20 column (Acclaim Pepmap C18 2 µm 100A, 75-µm i.d. x 15-cm length; Dionex) at a flow
21 rate of 300 nL.min-1.

22 Mobile phases were composed as indicated: A (99.9% water, 0.1% formic acid), B
23 (90% acetonitrile, 0.08% formic acid). The gradient consisted of a linear increase from
24 4% to 45% of B in 30 min, followed by a rapid increase to 70% within 1 min.
25 Composition was maintained at 70% B for 5 min and then decreased to 4% B for re-
26 equilibration of the column.

27 Mass data acquisitions were performed using Xcalibur 2.1 software. Full MS scans
28 were acquired at high resolution (FWMH 30,000) in the Orbitrap analyzer (mass-to-
29 charge ratio (m/z): 400 to 2000), while collision-induced dissociation (CID) spectra
30 were recorded on the five most intense ions in the linear LTQ traps.

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32 2.9. Database search and interpretation

33 Raw data collected during LC-MS/MS analyses were processed into mgf format files
34 and further searched against databanks using X!Tandem Spectrum Modeler. Protein
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identification was achieved by confronting mass data (MS and MS/MS spectra) against
four databases: (i) UniProt Knowledgebase (http://www.uniprot.org/) : restricted to
Viridiplantae (version 2011_03), (ii) Gene Index Databases Wheat release 12.0,
August 2010 (Wheat GI) (http://compbio.dfci.harvard.edu/tgi/ ), (iii) an in-house
glycosyltransferases databank (GTIDB) (http://wwwappli.nantes.inra.fr:8180/GTIDB)
[34], and (iv) a contaminant database including keratins and trypsin.

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A database search was performed with XTandem 2008.02.01
(http://www.thegpm.org/TANDEM/) via the X!tandem pipeline available at
http://pappso.inra.fr/bioinfo/xtandempipeline/. Enzymatic cleavage was declared as a
trypsin digestion with one possible miscleavage event. Fixed modifications of cysteine
residues by iodoacetamide as well as oxidized methionins were considered. Precursor
mass and fragment mass tolerance were set at 5 ppm and 0.8 Da, respectively. One
missed trypsin cleavage was allowed. Identified proteins were filtered according to the
following specifications: at least two peptides with an E value below 0.001 and a protein
E value below 10⁻⁴. To take redundancy into account, proteins with at least one peptide
in common were grouped.

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Protein identifications were compared in the three databanks. In the event of
identification in the three databanks, the identifier from UniProt will be preferentially
reported, followed by the one from Wheat GI and, finally, the one from GTIDB. The
search against the decoy database (Reverse Uniprot restricted to Viridiplantae) gave
a mean false-discovery rate of 0.3% for each query when using the p < 0.01
significance level.

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Tentative functional annotations were assessed by running a BLAST homology search
of sequences identified from mass spectrometry against the NCBI nr database using
Blast2GO (http://www.blast2go.org). The cut-off stringency for BLAST was E = 10⁻¹⁰.
For each protein, the best hit from each species was collected if present. The two sets
of proteins (protein sequences obtained from spectrometry analysis and their best
homologs) were analyzed by applying the Phobius (http://phobius.sbc.su.se/) and
TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) programs to predict
transmembrane domains (TM) and signal peptides.

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A refined search enabling protein identification from single peptide assignment was
performed to obtain more putative GTs. This extended protein list was then blasted
against the entire non-redundant sequences of the CAZy database using sequence
similarity or protein domains (Pfam) association rules [35]. Those for which an ortholog
1 was found in the CAZy database (e-value < 0.01) were reported. Proteins with
2 homology to GTs and GHs were listed, and when identified with only one peptide in
3 MS, the quality of the peptide was checked and validated manually.

4 For a subset of GTs and GHs, the best homolog (best Blast hit) in the rice and
5 *Brachypodium distachyon* genomes were searched using the Blast tool of the Rice
6 Genome Annotation website
7 (http://rice.plantbiology.msu.edu/analyses_search_blast.shtml). Nucleotide
8 sequences were translated with the ExPASy Translate Tool
9 (http://web.expasy.org/translate/) and amino acid sequences were aligned with
10 ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

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12 2.10 Transcriptomic analysis

13 A subset of GT and GH sequences were blasted against the sequence of the wheat
14 Affymetrix probe set using the PLEXdb Blast tool
15 (http://www.plexdb.org/modules/tools/plexdb_blast.php) [36]. The expression pattern
16 of the corresponding "genes" was retrieved from expression studies covering wheat
17 (whole) grain development [37] and different organs and tissues (PlaNet platform
18 WheatNet http://aranet.mpimp-golm.mpg.de/wheatnet) [38]. These analyses may not
19 discriminate all possible homologous copies within the wheat genome such as highly
20 similar paralogs and homoeologous forms of genes from the three genomes of
21 hexaploid wheat. Therefore, we will consider one "gene/transcript" when possibly
22 several forms co-exist.

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24 2.11. Subcellular localization

25 2.11.1 cDNA templates

26 Total RNA was isolated from manually dissected endosperm of wheat grains at 250°D.
27 About 1 g of tissue was ground in liquid nitrogen and extracted in 4.5 mL of buffer (0.1
28 M NaCl; 10 mM Tris HCl, pH 7.4, 1mM EDTA pH8; 1% SDS) with 3 mL phenol-
29 chloroform-IAA (25:24:1). After mixing and centrifugation, the supernatant was
30 extracted twice with 3 mL phenol-chloroform-IAA (25:24:1). The aqueous phase was
31 precipitated by the addition of 300 µL of sodium acetate (3M, pH 5.2) and 6 mL of
32 ethanol. RNA pellets were rinsed in 70% ethanol and dissolved in water. RNA was
33 treated twice with the DNase set (Qiagen) and then purified through the RNeasy
34 MinElute Cleanup kit (Qiagen), following the manufacturer's recommendations.
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Reverse transcription was carried out with 2 µg of total RNA from random hexamers using the Transcriptioner First Strand cDNA synthesis kit (Roche).

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## 2.11.2 Constructs

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All clones used in this study were constructed using Gateway technology (Invitrogen).

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The cDNA encoding the four GTs was amplified by PCR using a high fidelity KOD DNA polymerase (Novagen) with the primers listed in Supplemental Table 1. A two-step PCR procedure was carried out. A first PCR with gene-specific primers extended with part of the recombination regions attB1 and attB2 was achieved, and the resulting PCR products served as template for a second PCR, with the primers attB1 and attB2 for the GT47/Wheat GI ID: TC390543 and the GT61/UniProt ID: Q5QPY3 constructs, and the primers U5ATGSDK and U3 for the GT47/UniProt ID: E0ZPV1 and GT75/ Wheat GI ID: TC386312 constructs, to obtain the full recombination regions. Entry clones were obtained via BP reaction in pDONR-207 (Invitrogen). The binary pK7FWG2 and pK7RWG2 vectors containing the 35S promoter of cauliflower mosaic virus (CaMV) and the eGFP (enhanced Green Fluorescent Protein) or mRFP (monomeric Red Fluorescent Protein) respectively were used to construct C terminal fusions to the different GTs.

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## 2.11.3 Plant material and Agrobacterium-mediated transient expression

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*Nicotiana tabacum* sp. plants were cultivated in a controlled growth chamber at 21°C with 14h light/10h dark for 5-6 weeks prior to Agrobacterium infiltration.

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Transient expression of fluorescent protein fusions in tobacco leaf epidermal cells was performed as previously described by Sparkes et al. [39]. Briefly, *Agrobacterium tumefasciens* strains GV3101::mp90 were transformed by heat-shock with the binary plasmids. The transformants were then centrifuged and washed once with infiltration buffer (5 g/L glucose, 50 mM MES, 2 mM Na3PO4.12H2O, 100 µM acetonitrile) before being resuspended with the same buffer at an optical density (λ=600 nm) of 0.1 and 0.05 for GT constructs and Golgi markers (rat sialyl transferase signal anchor sequence (ST) fused to eGFP or mRFP), respectively. Lower leaves of *Nicotiana tabacum* sp. plants were infiltrated with the diluted bacteria using a syringe. For co-expression, the bacteria were mixed in appropriate volumes of infiltration buffer prior to injection into the leaves. Fluorescent protein expression was studied 2-3 days post-infiltration.

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2.11.4 Confocal laser scanning microscopy

3 Confocal microscopy experiments were performed at the facilities of the Biopolymers-
4 Structural Biology platform at INRA Nantes. An inverted Nikon A1 confocal laser
5 scanning microscope was used to examine the subcellular localization of GFP and
6 mRFP fluorescence. The samples were examined with a water-immersion x40
7 objective. GFP and mRFP channels were acquired by simultaneous scanning using
8 488-561 nm laser lines for excitation: emissions were collected via a photomultiplier
9 through band-pass filters, a 500-530 nm filter and a 570-620 nm filter. Images were
10 processed using NIS-Element Software (Nikon) and Adobe Photoshop 6.0.

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12 3. Results

13 3.1. Validation of the starting material and antibody used in the subcellular proteomic
14 approach

15 The wheat grain endosperm at 250°D was manually dissected and examined by
16 fluorescence microscopy, it contains the starchy endosperm as well as the aleurone
17 layer (Supplemental Fig. 1). At this stage, Golgi stacks are scarce compared to ER in
18 aleurone (Fig. 1A) and in starchy endosperm (Fig. 1B) cells, making the isolation of
19 fractions enriched in Golgi membranes challenging. Cell wall PATAg staining (Fig. 1C)
20 and AX immunolabeling (Fig. 1D) indicate that at this stage, cell wall polysaccharides
21 and AX, in particular, are actively produced in Golgi stacks and secreted into the wall.
22 The antibody used to detect Golgi membranes in our fractions was initially raised
23 against pea RGP1, a protein that has been localized in the Golgi cisternae of pea stem
24 subapical cells [12]. We checked the reactivity of this anti-RGP1 against wheat
25 endosperm. Immunogold labeling was detected in the Golgi apparatus of wheat
26 aleurone cells, validating the use of anti-RGP1 to detect Golgi apparatus-enriched
27 fractions (Fig. 2).

28

29 3.2. Subcellular fractionation

30 We developed a subcellular fractionation procedure to obtain fractions enriched in
31 Golgi apparatus from wheat endosperm. We first obtained a microsomal fraction that
32 was further fractionated into 15 subfractions that were characterized for their relative
33 content in Golgi stacks and ER. The fractions obtained in preliminary tests presented
34 a low organelle content, so we decided to pool the fractions obtained from four
1

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experiments. The results described in the next paragraphs were obtained from
approximately 120 g of endosperm isolated from wheat grains harvested between 250
and 275°C.

### 3.2.1. Generation of the microsomal fraction containing Golgi membranes

In order to obtain a sufficient amount of protein for characterization and proteomic
analysis from Golgi membrane-enriched fractions, six fractionations were conducted.

For each fractionation experiment, we obtained approximately 20 g of endosperm from
1200 wheat grains that were used to generate approximately 4 mL of microsomal
fraction by concentration on an iodixanol cushion.

The enrichment of these fractions in organelles was examined by Western blot
experiments performed with markers for two subcellular compartments, Golgi and ER
with RGP1 and BiP2, respectively (Fig. 3). Western blot analysis revealed that RGP1
protein was mainly present in the associated protein subfraction of the microsomal
interface, whereas BiP2 protein was only found in the membrane subfraction.

Accordingly, in the subsequent fractionation, the presence of BiP2 was only tested in
membranes and that of RGP1 in associated protein fractions (Fig. 3).

### 3.2.2. Generation of Golgi-enriched fractions

Microsomal fractions were further separated on a gradient generated from an iodixanol
solution of 14%, and 15 fractions were collected along the gradient. In each
experiment, the shape of the gradient was controlled by measuring the refractive index
of the fractions, and a good linearity was observed from densities between 1.05 and
1.1 (results not shown). The Western blots (Fig. 3) show the relative distribution of
Golgi stacks and ER along the gradient fractions. The good reproducibility of these
gradients led us to pool the fractions produced.

For protein identification, fractions revealing a high content in RGP1 and, at the same
time, a low content in BiP2, were selected (Fig. 3). Fractions 6, 7 and 8 were mixed in
one pool and referred to as G1, and fractions 11, 12, 13 were pooled and referred to
as G2. Both G1 and G2 were further fractionated into membranes and associated
proteins. The proteins extracted from the membranes pelleted from G1 and G2 will be
referred to as MPG1 and MPG2, whereas the associated proteins recovered from G1
and G2 will be referred to as APG1 and APG2. 1-D electrophoresis was used to reduce
the sample complexity; each electrophoresis gel was cut into 20 pieces before in-gel

20

21

22

23

24

25

26

27

28

29

30

31

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33

34
digestion and peptide extraction. These peptides were then analyzed by nano LC

MS/MS.

3

3.3. Protein identification

The sequencing of the wheat genome is underway. To identify proteins, we therefore

used UniProt restricted to Viridiplantae and, in parallel, the wheat GI database and

GT1db, an in-house database listing GT sequences from wheat, Arabidopsis and rice

[34], in which each protein is assigned to a GT family according to the CAZy

classification. Proteins identified from Wheat GI were annotated by running the BLAST
alignment algorithm [40] against the NCBI nr database.

10

11

3.3.1 Global analysis

First, a global analysis of all spectra generated from the four fractions was performed

by combining the result files corresponding to 81 bands cut in the previously described

electrophoresis experiment. This set of data generated a total of 1135 identified

proteins validated with at least two peptides, and all proteins with at least one common

peptide were grouped to avoid redundancy.

With these criteria, 668 proteins were selected (Supplemental Table 2), and the vast

majority of them were identified from the Wheat GI database. Their best homologous

proteins were then searched against the GenBank non-redundant protein database

(NCBI), and with the exception of one sequence, homologous proteins were found with

an e value < 10<sup>-10</sup>. Only 20% of the sequences were identified in wheat. The others

were identified by best homologs mainly found in *Hordeum vulgare* (54%), *Oryza sativa*

(15%), followed by other less represented species.

In a second analysis the spectra were grouped on the basis of the fraction they came

from, membranes (MPG) or associated (APG) Golgi proteins. Among the 668 proteins,

262 were found exclusively in membrane fractions (MPG1 and MPG2), 196 in the

associated protein fractions (APG1 and APG2) and 210 were found to be present in

both membranes and soluble fractions. These three sub-datasets were further

inspected for the presence of transmembrane domains (TM) and of signal peptides

(SP) using two different software programs, Phobius and TMHMM. TM and SP

predictions were examined in the sequences retrieved from the MS analysis as well as

in that of their best homologs. The output of these predictions [41] is reported in Table

1. Among the 262 sequences identified from the membrane fractions, 43% (112) had
1

2

3

4

5

6

7

at least one predicted TM domain. This proportion increased to 59% when the analysis was performed on their best homologs retrieved from the NCBI nr database. In the case of sequences identified exclusively in the APG fractions, the proportion of protein predicted with at least one TM drastically decreased to 4%, while among those found in both fractions, 14% were reported with at least one TM domain predicted.

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15

16

17

### 3.3.2 Search for carbohydrate-active enzymes

In order to identify all putative GTs and GHs, we ran BLAST search against the entire non-redundant sequences of the CAZy database by taking, as inputs, all identified protein sequences (1135) which returned a set of 78 sequences with a pfam domain (Supplemental Table 3). Our primary targets were the GTs, but many hits were found in the other classes of carbohydrate-active enzymes. In fact, we identified 29 putative GT homologs in 11 GT families. We also identified seven sequences for carbohydrate-binding modules, five of which also included a glycosyl hydrolase "GH13" domain. We therefore decided to assign them to the GH13 family. We thus identified 46 putative GH homologs distributed in 17 GH families. In addition, one sequence belonging to Ces was retrieved. It was similar to a pectin acetylesterase from the Ces13 family.

18

19

20

21

22

23

24

25

In order to identify all putative GTs and GHs, we ran a new BLAST search against the CAZy database based on similarity. This secondary search resulted in identifying six additional GT families (GT2, GT30, GT41, GT61, GT77 and GT92) and two GH families (GH2 and GH12) (Supplemental Table 3). In order to avoid missing low abundant proteins we also examined the GT and GH sequences retrieved by a BLAST search on proteins identified with a single peptide. Three of them were retained as they presented a peptide with a good quality score, reported at the end of Supplemental Table 2.

26

27

28

29

30

Finally, our search resulted in an increase in the number of families identified to 17 GT families and 19 GH families (Fig. 4). Further analysis revealed that GTs and GHs were identified in a specific fraction, e.g., either GH18, GT34 in MGP (in blue, Fig. 4) or GH77, GT75 in AGP (in red, Fig. 4). Surprisingly, some of them were found in both fractions.

31

32

33

Among the GT and GH families identified, the GT2, GT34, GT47, GT48, GT61, GT75, GH3, GH9 and GH17 were reported in the literature to contain members potentially involved in cell wall metabolism. The wheat sequences identified in these families were
1

assigned to their best BLAST hit in the genome of rice and *Brachypodium distachyon* and their putative or characterized functions are reported in Table 2.

2

3

4

## 3.4 Expression pattern of a subset of candidate genes

5

We analyzed the expression pattern of the subset listed in Table 2 by mining published and publicly available transcriptomic data. In Supplemental Fig. 2, the results show that several “genes” such as members of the GT34, GT61, GH3, GH9 and GH17 families exhibit a pattern of expression relatively specific to the grain. This result confirmed the importance of choosing the target tissue of interest as starting material for our analysis.

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12

## 3.5 Experimental validation of the Golgi localization for three chosen GTs

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30

31

To validate the Golgi localization of candidates identified with the subcellular proteomic approach, we chose GT families known for their potential involvement in cell wall biosynthesis. In particular, we selected GTs for which a complete nucleotide coding sequence was available at the time we did our analysis, and validated them *in silico* after nucleotide and amino acid sequence alignments with their best BLAST hit in rice and *Brachypodium distachyon*. The coding sequences corresponding to the GT75/Wheat GI ID: TC386312, GT47/UniProt ID: E0ZPV1, GT61/UniProt ID: Q5QPY3 (GenBank ID AK333763.1), and GT47/Wheat GI ID: TC390543 were inspected and the first three were found to be complete. In the case of GT47/Wheat GI ID: TC390543, sequence alignments with the best Brachypodium homolog and with wheat ESTs revealed a missing nucleotide in the coding sequence that shifted the reading frame (Supplemental Document 1). The *in silico* validated coding sequences of the three GTs were cloned using cDNA obtained from the endosperm of wheat grain at 250°C. The intracellular localization of the GT proteins was determined by transiently expressing them as fluorescent protein fusions in tobacco leaf epidermal cells. GT75/Wheat GI ID: TC386312, GT47/UniProt ID: E0ZPV1, GT61/UniProt ID: Q5QPY3, and GT47/Wheat GI ID: TC390543 were all expressed as small and mobile bodies in the cells, highly similar to the Golgi bodies. Co-expression of the GTs with a Golgi marker confirmed their localization in the Golgi apparatus (Fig. 5).

32

33

# 4. Discussion
The wheat endosperm cell wall composition is unusual and relatively simple, and appears to be a good model to reach enzymes implicated in the biosynthesis of their constituting polymers. It is thought that part of these enzymes and especially the GTs are located in the Golgi apparatus. Many of these GTs are membrane proteins present in low abundance; they were not detected in global proteomic analyses of the wheat grain endosperm [42-44]. The challenge of this work was to identify carbohydrate-active wheat enzymes at the proteomic level with a special focus on GTs potentially involved in the synthesis of cell wall polysaccharides in the grain endosperm. Additional difficulties had to be overcome. Only a few Golgi apparatus are visible in the starchy endosperm at 250°C. We chose this developmental stage because the endosperm is then easily isolated and because there is an active deposition of cell wall polysaccharides (in particular, the AX starts to accumulate), whereas grain filling with storage compounds (starch and storage proteins) is only beginning. Another difficulty lays in the fact that wheat proteins and especially wheat GTs are not well represented in protein databases.

We developed a procedure for the isolation of Golgi-enriched fractions adapted from that of Graham [45] and Sadowski et al. [46] followed by 1D protein analysis, in-gel digestion and mass spectrometry analysis of the resulting peptides.

## Isolation of Golgi-enriched fractions

The microsomal fraction we obtained revealed an enrichment in Golgi stacks and ER. The BiP2 was used as an ER marker and was only revealed in the membranes of our microsomal fraction, corroborating the results obtained by Mitra et al. [47] who identified BiP2 in the membrane sub-proteome of whole Arabidopsis seedlings.

RGP1 was used as a Golgi marker. We, however, detected RGP in both the soluble and the membrane subfractions of the microsome, which confirmed the presence of Golgi stacks in the microsomal fraction and corroborates previous results that report that RGPs have been found in both the membrane and soluble fractions of many species [48, 49]. A RGP was identified in the associated fraction (Wheat GI ID: TC386312) in our proteomic analysis.

Variation of the relative enrichment in Golgi or ER was observed along the gradient, allowing us to collect the most Golgi-enriched fractions for protein identification. The presence of BiP2 in these fractions can be either due to ER contamination or to a possible localization in Golgi as reported recently in Arabidopsis cell culture [50].
1 Proteomic analysis

2 The subcellular fractionation and LC-MS/MS analysis of wheat endosperm resulted in
3 the identification of 1135 unique proteins (at least two peptides with an E value < 0.001)
4 that, after grouping, gave 668 non-redundant proteins, 262 from the membrane
5 fractions and 196 from the associated fractions, meaning that 210 were found in both
6 fractions.

7 Membrane and associated proteins

8 Proteins only detected in the membrane fractions are expected to be strongly linked to
9 the membranes (integral membrane proteins and proteins anchored to the membrane),
10 whereas proteins within the membrane-associated fractions may be proteins in transit
11 within the endomembrane system and proteins linked to membrane proteins.

12 The proteins identified in both fractions may be due to a strong association between a
13 protein and a membrane protein within a complex where some but not all proteins are
14 integral membrane proteins as already described by Zeng et al. [28].

15 Furthermore, as recently reported by Atmodjo et al. [51], the transmembrane domain
16 of some type II proteins are cleaved in the Golgi apparatus, although the processed
17 proteins are retained in the Golgi apparatus within protein complexes. We cannot
18 exclude the possibility that for some of the proteins, the dual location may reflect a
19 contamination due to insufficient membrane washing, although the fractions were
20 extensively diluted (30 times) before membrane pelleting.

21 The prediction analysis of the transmembrane domain reflects this distribution: only
22 several percent of the proteins within the associated fraction are predicted to contain
23 a TM domain, whereas half of the proteins found only in the membrane fractions are
24 predicted to contain such a domain. This percentage would undoubtedly increase if the
25 wheat protein sequences were complete since many Golgi proteins have their
26 transmembrane domain in the N-terminal region.

27 Golgi localization

28 Many proteins transit via the endomembrane system (ER and Golgi) before being
directed to their final destination. In particular, secreted proteins undergo glycosylation
29 in the ER and the Golgi apparatus, and it has been postulated that about 50% of plant
30 proteins could be glycosylated [52]. Some proteins are retained in the Golgi apparatus
31 where they carry on their function.

32 In our Golgi-enriched fractions, we identified proteins that are not supposed to be Golgi
33 residents: abundant proteins (histones, ribosomal proteins, ribulose-1,5-bis-phosphate
34
1

carboxylase/oxygenase) that contaminate our fractions, as well as GTs such as the

2

callose synthase (Wheat GI ID: TC451164), most probably localized in the plasma

3

membrane and the starch synthase (UniProt ID: Q43654|SSY1_WHEAT) in plastids.

4

In addition to their transit in the Golgi apparatus, proteins can be recycled via the Golgi

5

apparatus. For example, the cellulose synthase and possibly the callose synthase,

6

both located in the plasma membrane, could be stored in the Golgi apparatus or

7

recycled to be directed where needed [50, 53]. In order to validate the Golgi location

8

of some of the GTs identified, four of them were co-expressed with a Golgi marker in

9

tobacco cells.

10

11

## Identification of proteins of interest

12

13

### Storage proteins

14

Our study focused on one specific developmental stage that corresponds to the very

15

early stage of storage deposition (starch and protein). The quality of our samples was

16

confirmed by the low amount of storage proteins found by our analysis. Less than 1%

17

of the identified proteins were storage proteins - five gliadins and three glutenins -

18

which are secretory proteins synthesized and folded into the ER before being

19

deposited into organelles known as protein bodies [54]. However, these proteins were

20

also detected in Golgi-derived vesicles early in the grain development, indicating that

21

the Golgi apparatus is also involved in prolamin transport [55, 56].

22

### Carbohydrate-active enzymes

23

Within the numerous proteins identified, it was predicted that 78 sequences would be

24

active on carbohydrates, among them, proteins from 17 GT families (Fig. 4).

25

Several of the GTs identified are clearly not involved in cell wall synthesis. GT4 and

26

GT5 are enzymes that catalyze the metabolism of sucrose (sucrose synthase UniProt

27

ID: Q8W1W4) and starch (starch synthase UniProt ID: Q43654). Interestingly, they

28

were only identified in the associated fraction. Members of the GT66 family (STT3

29

homolog GenBank ID: AAT69659) were implicated in protein N-glycosylation and

30

localized in the ER [23].

31

32

### GTs potentially involved in cell wall polysaccharide biosynthesis

33

Other GTs identified in our proteomic analysis have already been implicated in cell wall

34

synthesis. This is the case for the GT2 CsIF6 protein of wheat (Wheat GI ID:
1

TC390697). Homologs were identified as MLG glucan synthases following the insertion of rice *CsIF* genes into the *Arabidopsis thaliana* genome, which resulted in the detection of grass-specific MLG in the walls of the transgenic lines [57]. The wheat gene *CsIF6* was identified and silenced by RNA interference, resulting in a decrease of the MLG content in wheat grain endosperm [58].

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5

6

Conversely, we identified GTs known to be involved in AX synthesis. The GT47-13 protein UniProt ID: E0ZPV1_WHEAT was found in a complex with glucurono(arabino)xylan synthase activity in a wheat seedling microsome fraction [28].

7

8

9

Although no transmembrane domain is predicted for this protein, we localized it in the Golgi apparatus. The TaGT47-13 protein is the potential ortholog of Arabidopsis IRX10 and IRX10-L [59, 60], which have been implicated in the elongation of the xylan backbone.

10

11

12

13

RGP proteins are members of the GT75 family, although their role in glycosylation remains to be proven. Indeed, several recombinant rice and *Arabidopsis thaliana* RGPs were found to have a UDP-Arap mutase (UAM) activity, which is required to convert the UDP-arabinopyranose into UDP-arabinofuranose [61, 62]. Down-regulation of rice UAM reduces the proportion of arabinofuranose in rice cell walls [63].

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17

18

Zeng et al. [28] detected a GT75 protein (UniProt ID: Q9ZR33) in the glucurono(arabino)xylan complex that they isolated from a wheat seedling microsomal fraction. We identified three GT75 in the wheat endosperm (Wheat GI ID: TC386312, Wheat GI ID: TC371253, Wheat GI ID: TC399081), the sequence of Wheat GI ID: TC386312 is highly similar but not identical to UniProt ID: Q9ZR33. Our fusion experiment revealed that Wheat GI ID: TC386312 was localized in the Golgi apparatus, despite a lack of transmembrane domain.

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20

21

22

23

24

25

Very recently, several rice and wheat GT61 of the subgroup A were shown to be xylan O3 arabinosyltransferases (XATs) [19]. We identified three different GT61 of the subgroup A in the wheat endosperm (Table 2) and localized one of them, the UniProt ID: Q5QPY3 corresponding to XAT1, in the Golgi apparatus. Transcriptomic analysis showed that it seems specifically expressed in the endosperm. A GT61 from the subgroup B (UniProt ID: Q5QPZ5_WHEAT) was also found in our analysis (Table 2).

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27

28

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30

31

The corresponding gene exhibits an expression pattern specific to the grain endosperm and floral bracts before anthesis (Supplemental Fig. 2). This GT61 may be an arabinosyltransferase required for the double substitution of xylose residues in the

32

33
endosperm AX at position O2 and O3. It is noteworthy that no GT61 was identified in the glucurono(arabino)xylan complex isolated from wheat seedlings by Zeng et al. [28].

A GT48 protein was revealed in our analysis. The GT48 family is known to include the glucan synthase-like proteins (GSL) or callose synthase (CalS) [64, 65]. Callose plays an important role in plant development and in response to stress. Callose is deposited at cell plates during cytokinesis. It accumulates transiently in the wheat endosperm early in the development [4]. Voigt et al. [66] identified eight *GSL* genes in wheat and studied their expression in different organs. The protein we detected in the endosperm at this stage (Wheat GI ID: TC451164) corresponds to the *TaGSL12* gene.

It was very recently revealed that members of the CsID family have mannan synthase activity [67]. Mannans are present in the wheat endosperm. Nemeth et al. [58] produced RNA interference lines targeting the wheat *CsID2* gene potentially encoding the CsID2 protein that we detected by proteomic analysis (Wheat GI ID: TC394160).

They only investigated a potential effect on AX and MLG. Thus, it is possible that the wheat CsID2 has a mannan synthase activity. Virtually nothing is known about the structure, i.e., the possible substituents of mannans in wheat grain. Among the proteins we identified in our proteomic analysis, several GT34 share similarity with a galactomannan galactosyltransferase identified in lotus seeds [68]. The transcripts encoding the GT34 Wheat GI ID: TC370787 were detected in the grain but not in the vegetative organs investigated, and the transcript level increases steeply in the developing grain (Supplemental Figure 2). Mannans have a storage function in the seeds of lotus and many other species [69]. Nothing is known about their function in the wheat grain. Another activity was described for members of the GT34 family.

Several Arabidopsis genes of this family were found to encode xyloglucan xylosyltransferase [70-72]. Xyloglucan were recently detected transiently in the wheat endosperm [7].

Another interesting GT identified is the GT47 Wheat GI ID: TC390543 (best hit in rice LOC_Os11g03410), a protein containing a single transmembrane domain and that we localized in the Golgi apparatus. The GT47 family is encoded by 39 genes in Arabidopsis [73]. Phylogenetic analyses of the GT47 family revealed that LOC_Os11g03410 (and Wheat GI ID: TC390543) is in a different subgroup of GT47 (subgroup C) than GT47-13 (UniProt ID: E0ZPV1) and Arabidopsis IRX10-10L, which are in subgroup A according to the Zhong and Ye classification [7, 73]. Subgroup C contains several rice and Arabidopsis proteins. Only two of these proteins (ARAD1 and
1

ARAD2) have an ascribed function based on mutant characterization: they are putative
arabinosyltransferases acting on pectin [74]. Wheat GI ID: TC390543 and its rice
ortholog LOC_Os11g03410 are not really close to the ARAD proteins, and although
pectin is a component of the rice endosperm [75], no pectin has been reported in the
wheat grain endosperm [3]. Thus, it is possible that the enzyme encoded by Wheat GI
ID: TC390543 has a different activity.

2

3

4

5

6

7

We achieved our aim by identifying GTs potentially involved in wheat endosperm cell
wall synthesis through a proteomic approach.

8

9

10

GHs potentially involved in cell wall polysaccharide remodelling

11

Surprisingly, we also identified many GH proteins that we did not expect to be located
within the Golgi apparatus. To our knowledge, Golgi location was described for some
GHs, such as members of the families GH38 (Wheat GI ID: TC416977) and GH47
(Wheat GI ID: TC393888), which would be an alpha-mannosidase involved in protein
glycosylation [23, 50, 76]. It is thought that most of the other GHs are secreted. Many
GHs are glycosylated and probably pass through the Golgi apparatus. Half of these
GHs were identified in the associated fraction and only a few of them were predicted
to contain a TM domain, consolidating the idea that we identified them during their
transit through the endomembrane system.

12

13

14

15

16

17

18

19

20

The GH proteins identified in the endosperm fell into 18 of the 34 families that are
represented in the rice, Arabidopsis and Brachypodium genomes [77].

21

22

The putative function of some of the individual GHs was searched by studying the
annotation of their best homologs in other species. However, the occurrence within the
same family of numerous enzymatic activities makes the task of identifying the
potential activity of individual GHs difficult. Some GHs identified here are clearly not
involved in cell wall polysaccharide modification/turnover. In fact, the GH14 proteins
are beta-amylases (UniProt ID: Q7X9M2_WHEAT). Other families include enzymatic
activities that could modify cell walls.

23

24

25

26

27

28

29

The GH17 family contains many characterized activities, including β-1,3-glucanases
(EC 3.2.1.39) that specifically degrade callose, as shown in Arabidopsis
plasmodesmata [78]. We identified several GH17 in our experiment. BJ242908 is
similar to the rice putative β-1,3-glucanase (UniProt ID: Q9ZNZ1) and may be related
to the early and transient deposition of callose in the grain cell wall that is degraded
during development since callose is not detected in the mature grain endosperm. The

30

31

32

33

34
expression of the gene encoding GenBank ID: BJ242908 seems to be specific to the
grain endosperm and increases during grain development (Supplemental Fig. 2).
Fused with GFP, the callase identified in the plasmodesmata was localized in the
membrane and the ER [78].

Among the GH9, some were predicted to have an endo-β-glucanase activity. We
identified several GH9 sequences (GenBank ID: CD888519_WHEAT, Wheat GI ID:
TC382188, Wheat GI ID: TC442952). Best hit results by similarity search are endo-β-
glucanase (cellulase) implicated in leaf and fruit abscission in *Prunus persica* ppEG1,
*Citrus sinensis* and *Malus x domestica* [79]. In *Arabidopsis thaliana*, KORRIGAN is a
GH9 endo-β-glucanase that was proposed to play a role in cellulose synthesis [80-82].

The cell wall of wheat grain endosperm contains a minor amount of cellulose [2]. The transcript corresponding to Wheat GI ID: TC382188 seems to be specific to the grain endosperm and its expression increases during grain development (Supplemental Fig. 2).

Several GH3 were revealed in our analysis. Several activities were reported for
enzymes within the GH3 family. Among the GH3 identified in our screen, Wheat GI ID:
TC369308 is homolog to the barley β-D-glucan exohydrolase isoenzyme ExoII
(UniProt ID: Q42835_HORVU), and Wheat GI ID: TC422719 and Wheat GI ID:
TC384225 are homologs to a xylan β-D-xylosidase characterized in barley (Genbank
ID: AAK38482). Transcripts encoding the putative xylosidases are well represented in
the endosperm, and the abundance of Wheat GI ID: TC422719 increases considerably
during early development (Supplemental Fig. 2). Genbank ID: CK196562 is homolog
to the arabinofuranosidase/xylosidase identified in germinating barley (Lee et al. 2003).

The substrates of all these enzymes are most likely to be MLGs and AXs, the two main
components of the wheat endosperm cell wall. At the onset of their synthesis, the AXs
of wheat endosperm are highly substituted with arabinose and later in the endosperm
development, the level of AX substitution decreases [4, 5, 83, 84]. Toole et al. [84]
proposed that a GH with arabinofuranohydrolase or arabinofuranosidase activity would
remove arabinose residues to decrease the level of AX substitution. Recent studies
using films of pure AXs as models of endosperm cell walls revealed the influence of
AX structure on water diffusion [85, 86]. Higher water diffusion was obtained for films
made with highly-substituted AXs, compared to films made with lowly-substituted AXs.
This suggests that AX-decreased substitution may have a physiological function during
1

development, probably in the regulation of water/solute diffusion during grain
filling/dessication.

2

3

4

In summary, using this subcellular proteomic approach, we have successfully identified
over a thousand proteins from wheat starchy endosperm with many new enzymes
probably involved in cell wall biogenesis. They are members of families already
assumed to be involved in cell wall synthesis and modification or degradation, but
information about their function is still lacking. The expression pattern of the candidates
reveals that all of them were expressed in the wheat endosperm, with a few of them
exhibiting an expression profile specific to the endosperm. Synthesis of cell wall
hemicellulose takes place in the Golgi apparatus. We report here GTs potentially
involved in this mechanism, four of which were localized in the Golgi apparatus.

5

6

7

8

9

10

11

12

13

14

Acknowledgements:

15

16

This research was financed by the AIC and a CEPIA post-doc. We are grateful to
INRA Rennes for providing the wheat used in this experiment, and to G. Deshayes for
assistance with proteomics, P.E. Bodet and A. Geairon for assistance in mass
spectrometry and A. Bouder for her technical assistance. We thank our colleagues of
the Cell Biology Unit of the Imaging platform (Imagif) of the Institut des Sciences du
Végétal Gif sur Yvette, and of the "plateforme de Recherche en Imagerie Cellulaire de
Haute Normandie", Université de Rouen, in particular, Laurence Chevalier for their
help and support with sample preparation for ultrastructural and immunocytochemistry
TEM studies, respectively.

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<figure><img src="image_3.png" /><figcaption>Figure 1: Transmission electron micrographs of developing wheat grain at 250°D. A view of the cytoplasm showing different organelles in aleurone cells (A) and in the starchy endosperm (B). The aleurone cells exhibit dense granular cytoplasm with numerous vacuoles, ER and mitochondria. A few Golgi stacks are observed. In the starchy endosperm, ER tubules, some of which show dilation, are abundant, whereas Golgi structures with stacked cisternae are barely visible. PATAg staining of aleurone cell walls (C). PATAg-stained materials are detected within the cell walls and in Golgi-derived vesicles. Immunogold labeling of arabinoxylan in aleurone cells (D). Arabinoxylan epitopes are visualized in the cell wall. Labeling is mainly localized in the inner region of the primary cell walls and found over Golgi stacks.</figcaption></figure>

CW, cell wall; ER, endoplasmic reticulum; M, mitochondria; S, starch granule, V, vacuole; arrow, Golgi stacks.
<figure><img src="image_4.png" /><figcaption>Figure 2: Immunogold localization of RGP1 in aleurone cells. Gold particles occur over Golgi stacks but none over other cell structures.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Figure 3: Presence of Golgi apparatus and ER in microsomal fraction and fractions collected along the gradient revealed by western blot with antibodies against Golgi protein (Anti-RGP1 antibody) and ER protein (Anti-BiP2 antibody) as indicated. The anti-RGP1 results presented here were obtained on the associated protein fractions and those of Anti-BiP2 on membrane protein fractions.</figcaption></figure>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>Figure 4: Distribution of blasted sequences in (A) glycosyltransferase (GT) families and in (B) glycosylhydrolase (GH) families; number of hits obtained with the Pfam domain except for GTs and GHs labeled with a star for which the hits were obtained only by sequence similarity. Colors correspond to the fractions in which the sequences were identified: blue: present in membrane fractions; red: present in associated fractions; green: present in both fractions.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 5: The subcellular localization of some glycosyltransferases fused to fluorescent proteins in tobacco epidermal cells. Confocal images showing eGFP (green) and mRFP (red) fusion proteins coexpressed in tobacco leaf epidermal cells 2-3 days after agrobacterium infiltration. Images of cells expressing GT47-TC390543-eGFP (green) co-expressed with a Golgi marker, ST-mRFP (red) and merged image (A), GT75-TC386312-eGFP (green) co-expressed with ST-mRFP (red) and merged image (B), GT61-Q5QPY3-eGFP (green) co-expressed with ST-mRFP (red) and merged image (C), GT47-E0ZPV1-mRFP (red) co-expressed with ST-eGFP (green) and merged image (D). Scale bars = 10 µm.</figcaption></figure>
<table><caption>**Table 1:** Prediction of signal peptides (SP) and transmembrane domains (TM) using Phobius and TMHMM.</caption><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th rowspan="2"></th><th rowspan="2">Total number of sequences</th><th colspan="3">Phobius</th><th colspan="2">TMHMM</th></tr><tr><th>TM</th><th>SP</th><th>TM (%)</th><th>TM</th><th>TM (%)</th></tr></thead><tbody><tr><td rowspan="2">Membrane (MPG)</td><td>sequences</td><td></td><td>262</td><td>106</td><td>39</td><td>40.6</td><td>112</td><td>42.9</td></tr><tr><td>best Homologues</td><td></td><td>262</td><td>137</td><td>87</td><td>52.5</td><td>152</td><td>58.2</td></tr><tr><td rowspan="2">Associated (APG)</td><td>sequences</td><td></td><td>196</td><td>7</td><td>20</td><td>3.6</td><td>12</td><td>6.2</td></tr><tr><td>best Homologues</td><td></td><td>196</td><td>5</td><td>34</td><td>2.6</td><td>12</td><td>6.2</td></tr><tr><td rowspan="2">both fractions</td><td>sequences</td><td></td><td>210</td><td>30</td><td>36</td><td>14.3</td><td>35</td><td>16.7</td></tr><tr><td>best Homologues</td><td></td><td>210</td><td>35</td><td>63</td><td>16.7</td><td>35</td><td>16.7</td></tr></tbody></table>

<table><caption>**Table 2:** List of candidate GTs and GHs for a role in cell wall metabolism in the developing wheat endosperm</caption><thead><tr><th></th><th>proteomic hit</th><th>group</th><th>Families</th><th>best blast hit rice</th><th>E-value</th><th>annotation/putative function (rice)</th><th>brachypodium hit Activity in family and/or subgroup</th></tr></thead><tbody><tr><td>Wheat GI ID:</td><td>TC390697</td><td>359</td><td>GT2</td><td>LOC_Os08g08380</td><td>0</td><td>CSLF6 - cellulose synthase-like family F, beta1,3;1,4 glucan synthase</td><td>Bradifg25130 MLG synthase</td></tr><tr><td>Wheat GI ID:</td><td>TC394160</td><td>242</td><td>GT2</td><td>LOC_Os08g02180</td><td>0</td><td>CSLD2 - cellulose synthase-like family D</td><td>Bradifg50170 mannan synthase</td></tr><tr><td>Wheat GI ID:</td><td>TC370787</td><td>535</td><td>GT34</td><td>LOC_Os02g49140</td><td>1.0e-77</td><td>glycosyltransferase, putative</td><td>Bradifg56510 galactomannan galactosylTase</td></tr><tr><td>Wheat GI ID:</td><td>TC413183</td><td>958</td><td>GT34</td><td>LOC_Os11g34390</td><td>1.4e-75</td><td>glycosyltransferase, putative</td><td>Bradifg46670 galactomannan galactosylTase, xyloG xylosylTase</td></tr><tr><td>Wheat GI ID:</td><td>TC399543</td><td>444</td><td>GT47</td><td>LOC_Os11g03410</td><td>2.0e-209</td><td>exostosin family domain containing protein</td><td>Bradifg26150 subgroup C arabinosyltransferase acting on pectins</td></tr><tr><td>UniProt ID:</td><td>E0ZPV1</td><td>1091</td><td>GT47</td><td>LOC_Os01g70190</td><td>4.3e-219</td><td>exostosin family domain containing protein</td><td>Bradifg25940 subgroup A xylan xylosylTase (xylan synthase?)</td></tr><tr><td>Wheat GI ID:</td><td>TC451164</td><td>137</td><td>GT48</td><td>LOC_Os06g02280</td><td>8.2e-125</td><td>callose synthase, putative</td><td>Bradifg51760 callose synthase</td></tr><tr><td>UniProt ID:</td><td>Q5QPY3</td><td>289</td><td>GT81</td><td>LOC_Os02g22850.2</td><td>2.5e-184</td><td>glycosyltransferase, putative</td><td>Bradifg11300 XAT1 clade A xylan O3 arabinosyltransferase</td></tr><tr><td>Wheat GI ID:</td><td>TC437977</td><td>597</td><td>GT81</td><td>LOC_Os02g22650</td><td>1.5e-142</td><td>glycosyltransferase, putative</td><td>Bradifg11300 clade A xylan O3 arabinosyltransferase</td></tr><tr><td>UniProt ID:</td><td>D8L9S8</td><td>663</td><td>GT81</td><td>LOC_Os01g02930</td><td>3.5e-130</td><td>glycosyltransferase, putative</td><td>Bradifg01420 clade A xylan O3 arabinosyltransferase</td></tr><tr><td>UniProt ID:</td><td>Q5QFZ5</td><td>511</td><td>GT81</td><td>LOC_Os01g72610</td><td>6.4e-170</td><td>glycosyltransferase, putative</td><td>Bradifg61230 clade B unknown n function</td></tr><tr><td>Wheat GI ID:</td><td>TC386312</td><td>203.1</td><td>GT75</td><td>LOC_Os03g40270</td><td>1.1e-198</td><td>alpha-1,4-glucan-protein synthase, putative</td><td>Bradifg15050 RGPUDP-Arapmutase</td></tr><tr><td>Wheat GI ID:</td><td>TC371253</td><td>203.2</td><td>GT75</td><td>LOC_Os03g40270</td><td>5.1e-133</td><td>alpha-1,4-glucan-protein synthase, putative</td><td>Bradifg15050 RGPUDP-Arapmutase</td></tr><tr><td>Wheat GI ID:</td><td>TC399081</td><td>203.3</td><td>GT75</td><td>LOC_Os07g41380</td><td>1.3e-178</td><td>alpha-1,4-glucan-protein synthase, putative</td><td>Bradifg21990 RGPUDP-Arapmutase</td></tr><tr><td>Wheat GI ID:</td><td>TC384225</td><td>289</td><td>GH3</td><td>LOC_Os02g51620</td><td>2.3e-236</td><td>glycosyl hydrolase family 3 protein, putative</td><td>Bradifg59020 xylan B-D-xylosidase</td></tr><tr><td>Wheat GI ID:</td><td>TC422719</td><td>594</td><td>GH3</td><td>LOC_Os11g18730</td><td>1.2e-179</td><td>glycosyl hydrolase family 3 protein, putative</td><td>Bradifg42020 xylan B-D-xylosidase</td></tr><tr><td>Wheat GI ID:</td><td>TC389308</td><td>197</td><td>GH3</td><td>LOC_Os03g53800</td><td>1.2e-240</td><td>beta glucosidase</td><td>Bradifg08580 MLG endohydrolase</td></tr><tr><td>GenBank ID:</td><td>CK196562</td><td>1195</td><td>GH3</td><td>LOC_Os04g54810</td><td>0</td><td>beta-D-xylosidase, putative</td><td>Bradifg23470 arabinofuranosidase</td></tr><tr><td>GenBank ID:</td><td>CD888519</td><td>655</td><td>GH9</td><td>LOC_Os05g03840</td><td>1.8e-28</td><td>endoglucanase, putative</td><td>Bradifg38380 endo-1,4 beta-glucanase activity (cellulase)</td></tr><tr><td>Wheat GI ID:</td><td>TC382188</td><td>84</td><td>GH9</td><td>LOC_Os04g38610</td><td>1.4e-83</td><td>endoglucanase, putative</td><td>Bradifg611310 endo-1,4 beta-glucanase activity</td></tr><tr><td>Wheat GI ID:</td><td>TC442952</td><td>317</td><td>GH9</td><td>LOC_Os04g38610</td><td>2.1e-45</td><td>endoglucanase, putative</td><td>Bradifg611310 endo-1,4 beta-glucanase activity</td></tr><tr><td>GenBank ID:</td><td>BJ242908</td><td>641</td><td>GH17</td><td>LOC_Os01g71810</td><td>5.9e-59</td><td>glycosyl hydrolases family 17, putative</td><td>Bradifg260490 beta 1,3 glucanase callase</td></tr><tr><td>Wheat GI ID:</td><td>TC395632</td><td>237</td><td>GH17</td><td>LOC_Os01g71380</td><td>7.7e-86</td><td>glycosyl hydrolases family 17, putative</td><td>Bradifg260490 beta 1,3 glucanase callase</td></tr><tr><td>Wheat GI ID:</td><td>TC394973</td><td>466</td><td>GH17</td><td>LOC_Os01g71680</td><td>4.9e-37</td><td>glycosyl hydrolases family 17, putative</td><td>Bradifg260490 beta 1,3 glucanase callase</td></tr></tbody></table>