Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses

Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses Nadine Andersa, Mark D. Wilkinsonb, Alison Lovegroveb, Jacqueline Freemanb, Theodora Tryfonaa, Till K. Pellnyb, Thilo Weimara, Jennifer C. Mortimera, Katherine Stotta, John M. Bakerb, Michael Defoin-Platelc, Peter R. Shewryb, Paul Dupreea,1, and Rowan A. C. Mitchellb,1 a Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom; and Departments of bPlant Science and cBiomathematics and Bioinformatics, Rothamsted Research, Harpenden AL5 2JQ, United Kingdom

Edited* by Chris R. Somerville, University of California, Berkeley, CA, and approved December 7, 2011 (received for review September 27, 2011)

type II cell walls

| second-generation biofuels | dietary fiber

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ell walls provide shape and strength to different plant cell types and, moreover, constitute the majority of plant biomass. The cell wall composition of grasses, including the three most productive food crops, rice, wheat, and maize, and the energy crops miscanthus and sugarcane, diverged during evolution from dicots. A major distinguishing feature of grass cell walls is the prevalence and structure of the hemicellulosic component xylan (1). Xylan consists of a linear β-(1,4)-D-xylopyranose (Xylp) chain. It is most commonly substituted by arabinofuranose (Araf) on the C2- or C3-position in arabinoxylan (AX), and (4-O-methyl-) glucuronosyl side chains on the C2-position in glucuronoarabinoxylan (GAX) and glucuronoxylan (GX). The primary and secondary cell walls of grasses contain substantial amounts of GAX, which is also found in primary cell walls of dicots, but at much lower abundance (1, 2). In contrast, xylan in secondary cell walls of dicots is relatively abundant but devoid of arabinosyl side chains (2). The functional significance of the different side chains in planta is largely unknown. In grasses α-(1-3)–linked arabinofuranosyl substitutions can be esterified with p-coumaric or ferulic acid, the latter forming cross-links with other (G)AX chains (3) or with lignin (4). Cross-linking of cell-wall polymers is critical in limiting the digestibility of polysaccharides for bioenergy production and animal feed. In addition, AX has a role as dietary fiber in human foods, particularly in wheat flour, where it constitutes 65–70% of the nonstarch polysaccharide (5). The degree of arabinosylation and feruloylation of AX also determines whether it occurs as soluble or insoluble dietary fiber, which confer different benefits to human health (6). In Arabidopsis thaliana (Arabidopsis), several glycosyltransferases of the GT43 and GT47 families have been shown to be involved in the biosynthesis of the xylan backbone, including IRX9, IRX10, and IRX14 (2). The only enzymes characterized so far that decorate the xylan backbone are members of the GT8 family, GUX1 and GUX2, which are required for glucuronosyl substitution in Arabidopsis stem (7). www.pnas.org/cgi/doi/10.1073/pnas.1115858109

We previously used a bioinformatic approach to identify candidate genes involved in AX biosynthesis, reasoning that the greater abundance and different structure of xylan in grasses compared with dicots should be reflected in the expression of xylan biosynthesis genes. Orthologs of the xylan backbone synthesis machinery were identified, but GT61 genes were shown to have the greatest grass expression bias of all of the GTs (8). However, experimental evidence for the involvement of GT61s in xylan biosynthesis is lacking. Here we demonstrate that rice and wheat GT61 family members are responsible for α-(1,3)arabinosyl substitution of xylan, and thus name these proteins xylan arabinosyltransferases (XATs). Results Phylogenetic analysis of the GT61 enzymes showed that the family can be divided into three clades (Fig. S1A), of which only the divergent Clade C has been functionally characterized as Golgi-localized β-(1,2)-xylosyltransferases involved in N-glycosylation (9). Clade A is specific for higher plants with no representatives in Physcomitrella patens and Selaginella moellendorffii, and grass genes have undergone extensive duplication. Two Clade A GT61 genes, TaXAT1 and TaXAT2 (Fig. S1A), are abundantly expressed in wheat grain (10), which is consistent with their involvement in AX biosynthesis. Xylan is synthesized in the Golgi apparatus and XAT activity has been found in wheat Golgi membranes (11, 12). To determine whether our wheat XAT candidates are Golgi proteins, we expressed a GFP fusion of TaXAT2 (TaXAT2-GFP) in tobaccoleaf cells. TaXAT2-GFP fluorescence was detected in punctate structures that colocalize with the Golgi-marker GONST1 (13) (Fig. S1B), locating XAT to the site of AX biosynthesis. We have recently shown that six GT61 genes are expressed in wheat starchy endosperm and that TaXAT1 is much more highly expressed than any of the others, including the second most highly expressed, TaXAT2 (14). To investigate a role for GT61 genes in AX biosynthesis, RNAi constructs were therefore designed to suppress specifically TaXAT1 expression in the starchy endosperm of wheat. Although TaXAT2 is more expressed than TaXAT1 in whole-grain (10), in the starchy endosperm TaXAT2 expression is only about 12% of that of TaXAT1 (14) (Fig. S2A).

Author contributions: N.A., M.D.W., A.L., J.F., T.T., T.K.P., P.R.S., P.D., and R.A.C.M. designed research; N.A., M.D.W., A.L., J.F., T.T., T.K.P., T.W., J.C.M., K.S., and J.M.B. performed research; N.A., M.D.W., A.L., J.F., T.T., T.K.P., J.C.M., K.S., M.D.-P., P.R.S., P.D., and R.A.C.M. analyzed data; and N.A., P.D., and R.A.C.M. wrote the paper. Conflict of interest statement: The authors declare that a related patent application has been filed. *This Direct Submission article had a prearranged editor. Data deposition: The sequences reported in this paper have been deposited in the European Molecular Biology Laboratory database (accession nos. FR873610.1 and FR846232.1). 1

To whom correspondence may be addressed. E-mail: [email protected] or rowan. [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1115858109/-/DCSupplemental.

PNAS | January 17, 2012 | vol. 109 | no. 3 | 989–993

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Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by α-(1,2)– and α-(1,3)– linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases α-(1,3)–linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for α-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.

The TaXAT1 RNAi construct resulted in suppression of the endogenous TaXAT1 transcript by 6- to 30-fold, with no consistent effect on TaXAT2 (Fig. S2 B and C). Endosperm cell-wall fractions from RNAi lines were analyzed by digestion with a GH11 endo-xylanase followed by quantification of the resulting xylan oligosaccharides by high-performance anion-exchange chromatography (HPAEC). The ratios of oligosaccharide abundance in TaXAT1 RNAi transgenic samples compared with their azygous control are shown averaged across independent lines (Fig. 1A; see Fig. S3 for structures of all substituted xylan oligosaccharides). Interestingly, the abundances of all oligosaccharides containing monosubstituted α-(1,3)– linked Araf (XA3XX, XA3A3XX, XA3XA3XX, XA3A2+3XX, XA3XA2+3XX) were substantially decreased (Fig. 1A), and these decreases were also highly significant within all lines (Table S1). None of the other oligosaccharides were consistently affected, although a smaller effect on unsubstituted oligosaccharides was observed in four lines (Fig. 1A and Table S1). A decrease in total AX was confirmed by monosaccharide analysis in two of these lines (Table S2). Cell-wall fractions isolated from endosperm samples were further analyzed by 1H-NMR; this confirmed the results from HPAEC showing a specific decrease in the peak corresponding to H1 of Araf α-(1,3)–linked to monosubstituted Xylp in AX, whereas the peaks corresponding to H1 of Araf α-(1,2)– or α-(1,3)–linked to di-substituted Xylp were unchanged (Fig. 1B). The structural changes to AX induced by the transgene resulted in the water-extractable percentage of total AX being decreased from 42 to 18% in line 2, and from 33% to 21% in line 3 (Table S2).

A

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Fig. 1. Analysis of xylan structure in endosperm samples from homozygous TaXAT1 RNAi transgenic wheat. (A) Oligosaccharide abundance (HPAEC peak area) from transgenic samples relative to corresponding azygous controls after xylanase digest; mean of five independent lines ± 95% confidence intervals. Columns for AX oligosaccharides are colored according to substitution with Araf: unsubstituted (green), monosubstituted only (red), di-substituted only (blue), and mono- and di-substituted (purple). Abundances of (1,3);(1,4)-β-glucan oligosaccharides [Glucose3 (G3) and Glucose4 (G4)] as result of simultaneous lichenase digest show no overall change. (B) 1 H-NMR spectra for transgenic (red) and azygous control (blue) samples showing H1 signals for Araf in AX: α-(1,3)–linked to monosubstituted Xylp (A3-Xmono), α-(1,3)–linked (A3-Xdi), and α-(1,2)–linked (A2-Xdi) to di-substituted Xylp and for Araf in arabinogalactan peptide (A-AGP).

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These results in transgenic wheat strongly suggest that GT61 family members of Clade A are responsible for arabinosylation of xylan. To analyze the biochemical activity of GT61s in an in vivo system without intrinsic arabinosyltransferase activity, we chose heterologous expression of epitope-tagged wheat TaXATs in Arabidopsis stem (Fig. S4A). To mimic the situation in wheat endosperm more closely, we transformed the gux1 gux2 doublemutant (gux), which lacks glucuronosyl substitutions in stem xylan (7). Stem-derived alcohol-insoluble residue (AIR) was digested with GH11 endo-xylanase and the resulting fragments analyzed using polysaccharide analysis by carbohydrate gelelectrophoresis (PACE). In addition to the characteristic oligosaccharides generated by the xylanase digest of gux xylan (X, XX, XXX) (7), an additional minor fragment clearly detectable between marker (X)4 and (X)5 was released from the TaXAT2 AIR (Fig. 2A). Further digestion of the xylan oligosaccharides with arabinofuranosidase GH62, which specifically removes C2or C3-monosubstituted α-linked arabinofuranosyl substitutions (15), showed that the transgene-dependent oligosaccharide is arabinofuranosidase-sensitive (Fig. 2A). Given the migration in the gel and the specificities of the enzymes, we predicted the structure to have a degree of polymerization (DP) of 5, most probably being either XA3XX or XA2XX. To determine definitively the structure of the XAT-dependent xylan oligosaccharide, we used a modified oligosaccharide relative quantitation using isotope tagging (OliQuIT) method, which allows comparison of oligosaccharide abundances between samples by stable isotope tagging (16). Xylanase-digested AIR from TaXAT2-expressing gux transgenic plants was first compared with oligosaccharides similarly released from gux AIR, and second to TaXAT2-expressing gux transgenic material additionally digested with the arabinofuranosidase. Analysis by capillary normal-phase liquid chromatography (NP-LC) followed by MALDI-ToF-MS shows a single pentose oligosaccharide of DP5 in the XAT transgenic sample (Fig. 3 A and B). In contrast, structures of DP5 were not observed in the untransformed gux sample (Fig. 3A) or the XAT transgenic sample after arabinofuranosidase treatment (Fig. 3B), demonstrating that DP5 is both XAT-dependent and arabinofuranosidase-sensitive. The series of cross-ring 1,5X ions of the MALDI-ToF/ToF-MS/MS analysis showed that a pentose substitution is present on the penultimate xylosyl residue from the reducing end (Fig. 3C). The G3 and D2 elimination ions and the formation of the sugar lactone ion W2 show the presence of a pentose at the C-3 position of this Xylp (Fig. 3C). Taken together with the arabinofuranosidasesensitivity of the DP5 fragment, it can be unambiguously assigned as XA3XX. 1D 1H, 2D TOCSY and 13C HSQC NMR of extracted xylan from these plants confirmed that Araf α-(1,3)–linked to monosubstituted Xylp was present in the TaXAT2-transformed gux plants, but was not detected in untransformed gux plants (Fig. S4C). The signal strength suggests that the Ara substitution is present on ∼1% of the xylosyl residues, consistent with the low intensity of the oligosaccharide band by PACE. To determine whether the TaXAT2-activity is affected by glucuronic acid modifications on the xylan backbone and whether this function is conserved in other grass GT61 enzymes, we analyzed the xylan structure of further GT61-expressing transgenic lines. TaXAT2-expressing transgenic lines in the wild-type background also showed an additional xylan oligosaccharide not present in wild-type (Fig. 2B), and this oligosaccharide was arabinofuranosidase-sensitive (Fig. S5A). Tandem MS (Fig. S5B), together with knowledge of the xylanase and arabinofuranosidase specificities, identified the XAT-dependent oligosaccharide as XU4m2XXA3XX. We did not detect activity of TaXAT1 expressed in wild-type or gux backgrounds. However, we also analyzed FOX lines that express in Arabidopsis the cDNAs of two rice homologs of TaXAT2, OsXAT2 and OsXAT3, under the Anders et al.

Ta XA T2 wt Os XA T2 wt Os XA T3 wt

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CaMV 35S-promoter (17). The PACE fingerprint and MS analysis of both these lines also showed the presence of XU4m2XXA3XX (Fig. S5 A and C), and therefore indicates that these rice GT61 enzymes similarly direct the addition of α-(1,3)–linked Araf to the xylan backbone. Discussion As shown by RNAi suppression, TaXAT1 is responsible for the majority of monosubstitution of wheat starchy endosperm AX with α-(1,3)–linked Araf (Fig. 1). These results strongly suggest that grass XATs in GT61 Clade A possess xylan α-(1,3)-arabinosyltransferase activity, and this was confirmed by the heterologous expression of wheat and rice XAT genes in Arabidopsis. This activity is quite distinct from the previously described β-(1,2)-xylosyltransferase activity of N-linked glycan synthesis in GT61 Clade C. We found no evidence that α-(1,2)-, α-(1,3)-arabinosyl disubstitution is dependent on TaXAT1 activity, but the gene function was not entirely lost in the RNAi wheat lines. Therefore, the residual XAT activity could be sufficient to maintain α-(1,3)-arabinosyl substitution in disubstituted Xyl residues. On the other hand, the presence of the yet-uncharacterized GT61 Clade B enzymes and the multiplication of grass GT61s in Clade A might suggest distinct but related proteins are involved in α-(1,2)– and α-(1,3)–substitution of disubstituted Xyl residues. Indeed, the multiplicity of GT61 enzymes would permit a model where each enzyme requires subtly different substrates for arabinosylation, such as xylan already substituted with arabinosyl residues or acetyl groups. This finding could explain why the rice enzymes closely related to TaXAT2 were able to produce identical structure GAX oligosaccharides in transgenic Arabidopsis, whereas products of TaXAT1 were not detected. Members of the GT61 Clade A are also present in dicots. Expression levels of the Arabidopsis representatives, At3g18180 and At3g18170, are low and they are not coexpressed with secondary cell wall genes (Fig. S6). We found no evidence for arabinosylation of xylan in secondary walls in the absence of TaXAT2 Anders et al.

expression. This finding suggests that Arabidopsis GT61s are involved in α-(1,3)-arabinosylation of primary cell wall xylan. Although arabinosylation of xylan has yet to be demonstrated in Arabidopsis, it has been documented in seed mucilage (18) or primary walls (19) of other dicots. The reduction in Araf α-(1,3)–linked monosubstitution did not cause a concomitant increase in unsubstituted Xylp. This finding suggests there may be a requirement for substitution for continued xylan backbone synthesis in the wheat endosperm, which is unlike the gux xylan synthesis mutants of Arabidopsis, where xylan backbone synthesis is unaffected by the loss of GlcA substitution (7). Unlike acetylated GX synthesis in Arabidopsis, wheat endosperm xylan lacks acetyl substitution. Consequently, unsubstituted regions of xylan are likely to be insoluble. Indeed, the removal of monosubstitution is sufficient to cause a profound decrease in AX solubility presumably because of the greater hydrogen bonding between regions of AX chains lacking any substitution. Because feruloylation occurs via Araf α-(1,3)–linked to monosubstituted Xylp, a requirement of GT61 gene function for the feruloylation, and therefore cross-linking of xylan in grass cell walls, seems probable. Wheat endosperm has exceptionally low feruloylation of AX and GT61 involvement is best investigated in the future in tissues with substantial feruloylation. Changing the biosynthesis machinery that is required for the decoration of xylan by introducing genes characteristic for grasses into Arabidopsis provides a powerful approach for understanding gene function. The results also indicate potential applications; neither the Arabidopsis transgenics expressing wheat TaXAT2 in gux mutants (where we began to convert the usual dicot GX to AX) nor the wheat transgenics with drastically altered arabinosylation and solubility of AX, showed obvious effects on growth or development. This finding demonstrates the plasticity of xylan structure in planta. We have demonstrated a profound alteration of the properties of AX by manipulation of TaXAT1 in wheat flour, changing soluble fiber to insoluble fiber, so manipulation of GT61 activity promises to allow tailoring of AX biosynthesis to specific end-uses of cereal grain. PNAS | January 17, 2012 | vol. 109 | no. 3 | 991

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Fig. 2. PACE analysis of xylan structure of XAT transgenic material after digestion with xylanase. (A) Xylan fingerprint of gux in comparison with transgenics expressing TaXAT2 in gux background; +Arafase: additionally digested with arabinofuranosidase. Controls: Undigested material of gux without (1) and with (2) transgene. (B) Xylan fingerprint of wild-type (Wt) in comparison with transgenics expressing TaXAT2, OsXAT2 or OsXAT3 in wild-type background. Controls: Undigested material of wt (1), TaXAT2 wt (2), OsXAT2 wt (3), and OsXAT3 wt (4). Standard: Xylosyl oligosaccharides (X)1–6. Boxed area shows fivetimes longer exposure of identical gel. Asterisks mark the oligosaccharide specific for the transgenic lines. Oligosaccharide assignment is as follows. A, X; B, XX; C, XXX; D, XU(4m)2XX.

Phylogenetic Analysis. Substitutions and indels in GT61 coding DNA sequences were modeled with a maximum likelihood approach to generate the phylogenetic tree. Details are given in SI Materials and Methods.

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Fig. 3. Structural analysis of the arabinofuranosidase-sensitive xylan oligosaccharide derived from TaXAT2 gux. (A and B) Extracted ion chromatogram (EIC) of the sodiated molecule DP5 after xylanase digest. Capillary NPHPLC-MALDI-ToF-MS of AX oligosaccharide labeled with stable isotopes of aniline. The EICs of pentose DP5 labeled with 13C6-aniline (m/z 784) corresponds to TaXAT2 gux (blue) in comparison with labeling with 12C6-aniline (m/z 778; red) of (A) gux and (B) TaXAT2 gux after arabinofuranosidase digest (TaXAT2 gux +Arafase). (C) NP-HPLC-MALDI-ToF/ToF-MS/MS of the DP5 pentose XA3XX labeled with 2-AA (see Fig. S3 and ref. 25 for nomenclature). Ions mentioned in the text are highlighted in color. Note: G3, D2, and W2 ions show a α-(1,3)-linked pentose on the penultimate xylosyl residue.

Manipulation of GT61 genes could also provide a route to improve the digestibility of the vast quantities of lignocellulosic material from grass crops available for biofuel and animal nutrition.

Construct Preparation and Generation of Transgenic Material. The TaXAT2 and TaXAT1 full-length cDNA molecules were cloned from wheat var. Cadenza endosperm cDNA and sequenced (European Molecular Biology Laboratory accessions nos. FR873610.1 and FR846232.1, respectively). The TaXAT1 RNAi construct with the starchy endosperm-specific HMW1Dx5 promoter was created, wheat plants transformed and zygosity tested as described in ref. 20 and SI Materials and Methods. Homozygous and azygous (null segregants, used as within-line controls) T2 plants from five independent lines were grown in a statistical design and white flour samples were derived from mature T3 seed, as described in SI Materials and Methods. The pIRX9::TaXAT2-myc construct was comprised of intergenic regions from IRX9 to drive expression, the TaXAT2 coding sequence and Myc tag. Construct preparation, Arabidopsis transformation and Western blotting analysis are described in SI Materials and Methods. Analyses of Wheat Endosperm Cell Wall Composition. Preparation of endosperm cell-wall samples, enzymic digestion, and HPAEC analysis of resulting oligosaccharides were carried out following refs. 21 and 22, with modifications described in SI Materials and Methods. The amount of AX solubilized by this digestion (21) was estimated by monosaccharide analysis as described in ref. 23. Nonstarch polysaccharide from endosperm samples was prepared as described ref. 23 and used for monosaccharide and 1H-NMR analyses, as described in ref. 23 and SI Materials and Methods, respectively. Analyses of Arabidopsis Xylan. AIR was prepared, digested, and analyzed by PACE, as described in SI Materials and Methods. Determination of xylan structure was performed by 1H-NMR as described in SI Materials and Methods and a modified OliQuIT method and NP-LC–MALDI-ToF/ToF-MS/MS as follows. Five hundred micrograms to 1 mg AIR was alkaline-treated and digested as described in SI Materials and Methods and then dialyzed using Spectra/Por Biotech CE dialysis membrane MWC: 100–500D (Spectrum Laboratories) to remove excess of smaller fragments. For capillary NP-LC analysis using stable isotope tagging, dried-down samples were reductively aminated with light (12C6) or heavy (13C6) isotopes of aniline. Equal amounts of the differentially labeled samples were mixed, purified and analyzed by NPLC coupled offline to MALDI-ToF-MS, as previously described (16). This modified OliQuIT method ensures that the chromatographic elution positions on the same oligosaccharides labeled with different isotopes are identical, allowing accurate sample comparison. For structural analysis, samples were reductively aminated with 2-aminobenzoic acid (2-AA), purified and analyzed by NP-LC–MALDI-ToF/ToF-MS/MS, as previously described (24).

Details of plant material, reagents, PCR primers, quantitative RT-PCR, and localization experiments are described in SI Materials and Methods and Table S3.

ACKNOWLEDGMENTS. We thank Harry Gilbert and David Bolam for the gift of glycosylhydrolases and Steve Powers for statistical analyses. This work was supported by Biotechnology and Biological Sciences Research Council of the United Kingdom (BBSRC) Grant BB/F014295/1 (to R.A.C.M., P.R.S., and P.D.); and T.T. and J.C.M. were supported by BBSRC Sustainable Bioenergy Centre Cell Wall Sugars Programme Grant BB/G016240/1 (to P.D.). Rothamsted Research receives grant-aided support from the BBSRC. The research leading to these results received funding from European Community’s Seventh Framework Programme FP7/2007–2013 under Grant Agreement 211982 (RENEWALL).

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PNAS | January 17, 2012 | vol. 109 | no. 3 | 993

Supporting Information Anders et al. 10.1073/pnas.1115858109 SI Materials and Methods Generation of Phylogenetic Tree. All of the glycosyltransferase

(GT) 61 coding nucleotide and amino acid sequences present in Phytozome v7.0 (www.phytozome.net) were taken for the following fully sequenced organisms: Selaginella moellendorffii, Physcomitrella patens, Arabidopsis thaliana, Populus trichocarpa, Oryza sativa, and Sorghum bicolor. Proteins were identified as GT61 family by being in PFAM PF04577 or having KEGG Ortholog identifier K03714, which gave the same set of Arabidopsis proteins listed as GT61s at CAZy [www.cazy.org; (1)]. Together with coding regions of the wheat sequences TaXAT1 and TaXAT2 sequenced here, amino acid sequences were aligned using three multiple sequence alignment (MSA) methods, namely Muscle (2), MAFFT (3), and ClustalW (4). The MSA having the best identity score was first identified and then automatically trimmed using the software trimAL (5) (default “automated” setting). The resulting amino acid MSA was then back-translated codon-by-codon to form a nucleotide MSA and used to carry out a statistical selection of nucleotide substitution models with jMODELTEST (6). The best substitution model found according to the Akaike Information Criterion criterion was the GTR +G+I. A phylogenetic reconstruction has been conducted using the suite of software BEAST (7). First, a parameter file with the GTR+G+I model, an uncorrelated relaxed clock (mean fixed to 1.0) and a birth-death prior was set up. This parameter file was then passed to BEAST and three runs were performed (each Markov-chain Monte Carlo chain was 50-million steps long and was sampled every 5,000 steps). After we checked the mixing and the convergence of the chains with Tracer (6), an estimate of the phylogenetic tree was obtained using the consensus method in TreeAnnotator (6) (using burn-in fixed to 10%, maximum clade credibility and mean node heights). An unrooted version of the resulting consensus tree, with branch length in units of substitutions per site, is shown in Fig. S1A. Plant Material and Growth Conditions. Wheat plants were grown in temperature-controlled glasshouse rooms, as previously described (8). Arabidopsis seeds were surfaced-sterilized and then sown on solid medium [0.5× Murashige and Skoog salts, 1% sucrose (wt/ vol), pH 5.8] and, if applicable, selected by addition of 15 μg/mL of hygromycin or 30 μg/mL of phosphinothricin. After stratification for 48 h, plates were transferred to the growth room (20 °C, 16-h light/8-h dark) and after 1–2 wk the seedlings were transferred to soil. The Fox line for OsXAT2 (LOC_Os02g22480, K36913) and for OsXAT3 (LOC_Os03g37010, K06419) were developed by the Rice Genome Project of the National Institute of Agrobiological Sciences and provided by the Rice Genome Resource Center, Japan (9). Construct Preparation and Generation of Transgenic Material. The

TaXAT1 (TaGT61_1) and TaXAT2 (TaGT61_2) full-length cDNAs were cloned from wheat var. Cadenza endosperm cDNA using primer pairs GT61-1F1, GT61-1R1 and GT61-2F3, GT612R, respectively (Table S3) cloned into pGem-T Easy system (Promega) and sequenced (European Molecular Biology Laboratory accession nos. FR873610.1, FR846232.1). Two variants (putative homeologues from the three genomes of hexaploid wheat) of each sequence were also found in this cDNA sample, these variants had >96% nucleotide identity to TaXAT1 or TaXAT2. The TaXAT2 coding sequence was fused in frame to Anders et al. www.pnas.org/cgi/content/short/1115858109

the 5′ end of GFP5 in pVKH18-En6 as described in ref. 10, using primer GT61-2XbaF and GT61-2SalR (Table S3) to introduce unique XbaI and SalI restriction sites at the 5′ and 3′ end, respectively. The resulting TaXAT2-GFP construct was used for transient expression in tobacco. The TaXAT1 RNAi construct with the starchy endospermspecific HMW1Dx5 promoter was created as described in ref. 8, except that here the target fragment 235–729 bp of the 1,521-bp TaXAT1 sequence was used. This fragment has identical stretches of >130 bp with the two variants of TaXAT1, but the longest stretch of identity with any TaXAT2 transcript is 16 bp; thus, it would be expected to suppress all forms of TaXAT1 and have no effect on TaXAT2. Wheat plants were transformed and zygosity was determined in T1 seeds as previously described (8). Five independently transformed plants carrying the RNAi transgene were identified by PCR and homozygous and azygous (null segregants, used as within-line controls) T1 progeny were identified by quantitative PCR. These five plants were the only lines identified which had both homozygous and azygous progeny and there was no selection of lines by phenotype. T2 plants were grown in a complete four-block statistical design with four replicate pots per line (four plants per pot) and one pot per block. Mature T3 seeds were harvested and pooled together for each pot to give 10 g of seeds for each of the four biological replicate samples per line. The moisture of the samples was measured using a near-infrared spectroscopy (NIRFlex N-500; BÜCHI) and adjusted to 15%. These seed samples were milled in a Micro Scale LabMill (FQC-2000; Metefém Szövetkezet) and passed through 250-μm and 150-μm sieves to give pure white flour from the starchy endosperm. The pIRX9::TaXAT2-myc construct used for stable transformation of Arabidopsis consists of the PCR-amplified 1923bp 5′ intergenic genomic region of IRX9 (At2g37090; primer KpnI At2g37090 and ApaI At2g37090) (Table S3). The fragment was cloned into pGreen [BASTA resistance (BAR)] with KpnI and ApaI restrictions sites. A 1,500-bp 3′ intergenic genomic region of IRX9 was amplified using primer SmaI At2g37090 and NotI At2g37090 (Table S3) and then cloned in via SmaI and NotI restriction sites. The PCR-amplified wheat cDNA of TaXAT2 without stop codon (primer ApaI TaXAT2 and SmaI XhoI TaXAT2) (Table S3) was inserted using ApaI and SmaI restriction sites, adding a XhoI restriction site with the antisense primer. A 3× Myc tag was then inserted via the XhoI restriction site. The complete modified area was sequenced and the vector transformed into Arabidopsis wild-type (ecotype: Columbia) and gux1-2 gux2-1 (gux) double-mutants (At3g18660, SALK_063763; At4g33330, GABI_722F09) (11) using the floral dipping technique (12). At least three independent transgenic lines were analyzed and data of a representative line are shown. Quantitative RT-PCR of TaXAT Transcripts in Wheat Endosperm. Developing T3 seeds were harvested at 17 d post anthesis and pure starchy endosperm from the central 16 grains was dissected using a razor blade, snap-frozen in liquid nitrogen, and lyophilized. Following homogenization in a TissueLizer (Qiagen), total RNA was isolated according to ref. 13, and adjusted to microfuge scale. cDNA synthesis and qRT-PCR was performed as described in ref. 14 with the exception of using the Invitrogen Platinum Green qPCR SuperMix-UDG (www.invitrogen.com). Expression of endogenous TaXAT1 was determined with a 139-bp amplicon (primers prTYW13 and prTYW14) (Table S3) and TaXAT2 expression was determined with a 100bp amplicon (primers 1 of 10

prTYW412 and prTYW413) (Table S3). (These primers are all designed to regions of TaXAT1 and TaXAT2, which are identical in the three forms found and will therefore determine total transcript abundance for all forms.) Three reference genes were used to normalize expression: Ta2526, a stably expressed EST from grain (primer prTYW19 and TYW20) (Table S3), GAPDH (primer prTYW422 and prTYW423) (Table S3), and Succinate dehydrogenase (SDH, primer prTYW424 and prTYW425) (Table S3). Data were analyzed according to ref. 15. Because the TaXAT1 and TaXAT2 amplicons are of similar length, located at similar positions from the 3′ end of the respective target transcripts, and show almost identical individual PCR efficiencies (1.89 and 1.88), they can be used to compare expression of TaXAT1 and TaXAT2 in the same sample. This process was done for azygous controls, and ratio of expression values in transgenic samples relative to their azygous control samples for each of TaXAT1 and TaXAT2 also determined. Transient Expression of Proteins in Tobacco and Confocal Analysis.

TaXAT2-GFP was transformed into Agrobacterium tumefaciens (GV3101) to transform the lower epidermal surface of Nicotiana tabacum, as previously decribed (16). The GONST1-YFP construct (17) was coinfiltrated. Confocal imaging was performed using an inverted laser scanning microscope (Zeis LSM510 META) with a 63× oil immersion objective. For imaging expression of GFP and YFP, excitation lines of an argon ion laser of 458 nm for GFP and 514 nm for YFP with a 475/525-nm bandpass filter for GFP and a 530/600-nm band-pass filter for YFP were used alternately with line-switching, using the multitrack facilities of the microscope. Appropriate controls were performed to exclude any cross-talk and bleed-through of fluorescence.

1

H-NMR Analyses of Wheat Endosperm. Nonstarch polysaccharide from transgenic and azygous control endosperm (white flour) samples was prepared as previously described (21) and suspended in D2O [containing 0.01% (wt/vol) d4-TSP] at a concentration of 1 mg/mL. NMR spectra were recorded at 70 °C on a Bruker DPX 400 spectrometer (Bruker Biospin) using 512 scans of 64 K datapoints over a sweep width of 24 ppm, the residual water peak was suppressed by presaturation. AX peak assignments were based on (22, 23): H1 Araf δ peaks at 5.39 ppm for Araf α-(1,3)–linked to monosubstituted nonterminal Xylp and 5.28 ppm and 5.21 ppm for Araf α-(1,3)–linked and α-(1,2)–linked to di-substituted nonterminal Xylp, respectively. [The last value takes account of a change of −0.02 ppm from references above for instrument conditions used here (24)]. Arabinogalactan peptide (AGP) H1 Araf peak at 5.25 assignment is based on ref. 25 and confirmed under these conditions by spiking samples with AGP preparations (26). Transgenic sample and azygous control from two independent lines were analyzed and spectra from a representative line (line 3) are shown. Preparation of Alcohol-Insoluble Residue. Basal inflorescence stems from 5-to 6-wk-old plants were boiled for 30 min at 70 °C in 96% (vol/vol) ethanol and homogenized using a ball mixer (Glen Creston). The pellet was successively washed with 100% (vol/vol) ethanol, chloroform:methanol 2:3 (twice), 65% (vol/vol), 80% (vol/vol), and 100% ethanol and then air-dried. Enzyme Hydrolysis and Analysis of Oligosaccharides by Polysaccharide Analysis by Carbohydrate Gel Electrophoresis. One hundred mi-

Analysis of Arabinoxylan and (1,3);(1,4)-β-Glucan by High-Performance Anion-Exchange Chromatography. Analysis of endosperm arabi-

crograms of alcohol-insoluble residue (AIR) was treated with 4 M NaOH for 1 h, and neutralized with HCl. Xylan was digested in 0.1 M ammonium acetate buffer (pH 5.5) overnight at room temperature with excess of enzyme to ensure complete digestion. Enzymes used were: endo-xylanase GH11, NpXyn11A from Neocallimastix patriciarum (27) and α-arabinofuranosidase GH62, Abf62A from Pseudomonas cellulosa (28), both kind gifts from H. Gilbert (Newcastle University, Newcastle upon Tyne, United Kingdom). Xylan standards (X)1–6 were purchased from Megazyme. After digestion, samples were lyophilized in a centrifugal vacuum evaporator, labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and 2% of starting material analyzed by PAGE, as previously described (29). ANTS-signals were visualized using a Genebox (Syngene) equipped with a transilluminator with long-wave tubes emitting at 365 nm and a short-pass (500– 600 nm) filter.

noxylan (AX) and (1,3);(1,4)-β-glucan by digestion with endoxylanase (Xylanase M1; Megazyme) and lichenase (Megazyme) followed by high-performance anion-exchange ehromatography (HPAEC) of resultant oligosaccharides was as described in ref. 8, with the exception that the column used was Carbopac PA-1 (Dionex) with dimensions 2 mm × 250 mm and the flow rate was 0.25 mL/min. Peaks were identified from the retention times of reference compounds established during the development of this technique (19, 20). Resulting peak areas are a measure of abundance per unit dry weight for a specific oligosaccharide. Each of the four biological replicates was analyzed in three technical repeats. ANOVA was applied to oligosaccharide abundances, taking account of the randomized block design and biological and technical replication. The Cadenza wild-type line was included, giving an overall control plus five-by-two factorial treatment structure. Main effects of transgene (n, azygous null control; H, homozygous transgenic), line (1–5) and interaction were tested. (Tables S1 and S2). To summarize the effect of the transgene over all lines (Fig. 1A), ratios of mean oligosaccharide abundance in transgenic samples to that in their azygous controls were averaged across all lines.

Arabidopsis AIR Fractionation and Analysis by NMR. AIR (200 mg) was chemically fractionated according to ref. 30, and the 1 M KOH (xylan-rich) fraction lyophilised following extensive dialysis against water. The 1 M KOH fraction or wheat arabinoxylan (10 mg) was digested with endoxylanase GH11, as described above. Samples were lyophilised, resuspended in D2O (700 μL; 99.9% purity; Cambridge Isotope Labs) and transferred to a 5-mm NMR tube. NMR spectra were recorded at 298 K with a Bruker DRX spectrometer operating at 500 MHz equipped with a TXI probe. Chemical shifts were measured relative to internal acetone at δ= 2.225 ppm. Two-dimensional NOESY, TOCSY, and 13 C-HSQC were recorded using established methods (31); the mixing times were 70 ms and 200 ms for the TOCSY and NOESY, respectively. Data were processed using the Azara suite of programs (v. 2.8, copyright 1993–2011, Wayne Boucher and Department of Biochemistry, University of Cambridge, United Kingdom) and chemical-shift assignment was performed using Analysis v2.1 (32). Assignment of Araf substituents was assisted by the endoxylanase GH11 digested wheat arabinoxylan sample, and published chemical-shift data (23).

Protein Extraction and Western Blotting. Basal inflorescence stem material from 5- to 6-wk-old plants was ground in 1× Laemmlibuffer (60 mM Tris/HCl pH6.8, 2% (wt/vol) SDS, 10% (vol/vol) Glycerin, 0.01% Bromophenol Blue, 1.5% (vol/vol) β-Mercaptoethanol), boiled for 3 min, and centrifuged to remove cell debris. SDS/PAGE and protein gel blotting were performed as previously described (18). Antibody dilutions were: c-Myc (A14) antibody (Santa Cruz), 1:1,000, goat anti-rabbit antibody conjugated to horseradish peroxidase (Bio-Rad), 1:5,000. Detection was performed with the Amersham ECL Plus Western Blotting Detection System (GE Healthcare).

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1. Cantarel BL, et al. (2009) The carbohydrate-active enzymes database (CAZy): An expert resource for Glycogenomics. Nucleic Acids Res 37(Database issue):D233–D238. 2. Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. 3. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30: 3059–3066. 4. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. 5. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T (2009) trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972–1973. 6. Posada D (2008) jModelTest: Phylogenetic model averaging. Mol Biol Evol 25:1253–1256. 7. Drummond AJ, Rambaut A (2007) BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 7:214. 8. Nemeth C, et al. (2010) Down-regulation of the CSLF6 gene results in decreased (1,3; 1,4)-beta-D-glucan in endosperm of wheat. Plant Physiol 152:1209–1218. 9. Kondou Y, et al. (2009) Systematic approaches to using the FOX hunting system to identify useful rice genes. Plant J 57:883–894. 10. Dunkley TPJ, et al. (2006) Mapping the Arabidopsis organelle proteome. Proc Natl Acad Sci USA 103:6518–6523. 11. Mortimer JC, et al. (2010) Absence of branches from xylan in gux mutants reveals potential for simplification of lignocellulosic biomass. Proc Natl Acad Sci USA 107: 17409–17414. 12. Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana. Plant J 16:735–743. 13. Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116. 14. Pellny TK, et al. (2008) Mitochondrial respiratory pathways modulate Arabidopsis nitrate sensing and nitrogen-dependent regulation of plant architecture in Nicotiana sylvestris. Plant J 54:976–992. 15. Rieu I, Powers SJ (2009) Real-time quantitative RT-PCR: Design, calculations, and statistics. Plant Cell 21:1031–1033. 16. Brandizzi F, Snapp EL, Roberts AG, Lippincott-Schwartz J, Hawes C (2002) Membrane protein transport between the endoplasmic reticulum and the Golgi in tobacco leaves is energy dependent but cytoskeleton independent: Evidence from selective photobleaching. Plant Cell 14:1293–1309. 17. Baldwin TC, Handford MG, Yuseff MI, Orellana A, Dupree P (2001) Identification and characterization of GONST1, a golgi-localized GDP-mannose transporter in Arabidopsis. Plant Cell 13:2283–2295.

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18. Lauber MH, et al. (1997) The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J Cell Biol 139:1485–1493. 19. Ordaz-Ortiz JJ, Devaux MF, Saulnier L (2005) Classification of wheat varieties based on structural features of arabinoxylans as revealed by endoxylanase treatment of flour and grain. J Agric Food Chem 53:8349–8356. 20. Saulnier L, et al. (2009) Wheat endosperm cell walls: Spatial heterogeneity of polysaccharide structure and composition using micro-scale enzymatic fingerprinting and FT-IR microspectroscopy. J Cereal Sci 50:312–317. 21. Englyst HN, Quigley ME, Hudson GJ (1994) Determination of dietary fibre as nonstarch polysaccharides with gas-liquid chromatographic, high-performance liquid chromatographic or spectrophotometric measurement of constituent sugars. Analyst (Lond) 119:1497–1509. 22. Hoffmann RA, Geijtenbeek T, Kamerling JP, Vliegenthart JF (1992) 1H-N.m.r. study of enzymically generated wheat-endosperm arabinoxylan oligosaccharides: Structures of hepta- to tetradeca-saccharides containing two or three branched xylose residues. Carbohydr Res 223:19–44. 23. Gruppen H, et al. (1992) Characterisation by 1H NMR spectroscopy of enzymically derived oligosaccharides from alkali-extractable wheat-flour arabinoxylan. Carbohydr Res 233:45–64. 24. Toole GA, et al. (2010) Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward. Planta 232:677–689. 25. Loosveld AMA, Grobet PJ, Delcour JA (1997) Contents and structural features of water-extractable arabinogalactan in wheat flour fractions. J Agric Food Chem 45: 1998–2002. 26. Tryfona T, et al. (2010) Carbohydrate structural analysis of wheat flour arabinogalactan protein. Carbohydr Res 345:2648–2656. 27. Vardakou M, et al. (2008) Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases. J Mol Biol 375:1293–1305. 28. Beylot MH, McKie VA, Voragen AG, Doeswijk-Voragen CH, Gilbert HJ (2001) The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity. Biochem J 358:607–614. 29. Goubet F, Jackson P, Deery MJ, Dupree P (2002) Polysaccharide analysis using carbohydrate gel electrophoresis: A method to study plant cell wall polysaccharides and polysaccharide hydrolases. Anal Biochem 300:53–68. 30. Brown DM, et al. (2007) Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis. Plant J 52:1154–1168. 31. Cavanagh J, Fairbrother WJ, Palmer AG, Skelton NJ (1996) Protein NMR Spectroscopy: Principles and Practice (Academic Press, San Diego, CA). 32. Vranken WF, et al. (2005) The CCPN data model for NMR spectroscopy: Development of a software pipeline. Proteins 59:687–696.

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Sb04g 02021 0 Os02g22190 Sb 03g 007 900 Sb04g02850 Os0 OSs b 1 Os0 2g04250 02 0 g 0 6g4 g2 2 9 932 0 2 3 T a 650 8 0 XA T1

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124 Os06g28 100 g 0 3 5 10 S b 0 4 4g120 20 2 0 9 Os0 g02 0 7 90 01 g 0 5 Os b 0 3 079 S g0 03 Sb

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0 0 80 0 2 02 3 5 g0s10g 20 0 1 O 28 00 S b 3g04 A T 3 1 8 3 T 2 0 0 A S b OsX 1 0 g T a X Sb b04g013150 S OsXAT2 At3g18180 At3g1 8170 Poptr_0015s04180 Poptr_0233s00200 Pop tr_0 Poptr_0 012s Sb 09 g0 19 0484 015s04170 36 0 Os05 0 g 3 254 S O Osb 0 2 gs01g3 0 07 0 4 1370 S b g46 1 6 5 0 0 3 38 Sbg0 0 0 0 3 911 g0 0 46 29 0

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Clade B

0.2

B

composite

GONST1-YFP

TaXAT2-GFP

Fig. S1. Phylogenetic tree of GT61 family and subcellular localization of xylan arabinosyltransferases (XAT) protein. (A) Phlyogenetic tree. Lower plants are represented by S. moellendorffii (dark red) and P. patens (orange); monocots by O. sativa (dark green), S. bicolor (light green), and the two wheat genes TaXAT1 and TaXAT2 (black); and dicots by A. thaliana (dark blue) and P. trichocarpa (light blue). Asterisks mark proteins studied in this report. Note: Proteins of Clade A and B contain the DUF563 domain (PFAM family PF04577), which is not present in proteins of Clade C. (B) Confocal analysis of TaXAT2-GFP (green), Golgi-marker GONST-YFP (red) in tobacco epidermal cells, and overlay of both (yellow).

0.5

TaXAT1 TaXAT2

0.4 0.3 0.2 0.1 0

EExpression transggenic/control

B 0.6

1

TaXAT2

0.8

2 1.5

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Line 1 Line 2 Line 3 Line 4 Line 5

C TaXAT1

Expression trransgenic/contro ol

Relative Gene Expression in controls

A

0 Line 1 Line 2 Line 3 Line 4 Line 5

Line 1 Line 2 Line 3 Line 4 Line 5

Fig. S2. Quantitative RT-PCR determination of TaXAT1 and TaXAT2 transcript abundance in wheat starchy endosperm. (A) Average gene expression ± SE of TaXAT1 and TaXAT2 in the azygous control samples from the five lines. (B and C) Average ratio of expression in homozygous TaXAT1 RNAi transgenic samples relative to their azygous controls ± SE for TaXAT1 (B) and TaXAT2 (C).

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XA3XX DP5

O

HOCH2

HO HO

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O

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OH

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OH

OH

HO

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O

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O

OH

OH

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OH

OH

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HO

OH

O

OH

H3CO/HO OH

OH

XA2+3XX DP6

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HOCH2

HO HO

O O

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O

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O

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O

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OH

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OH

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HOCH2

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O

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OH

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OH

O

HOCH2

HO

HO

OH

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OH

XA3XA3XX DP8

O

O O

OH

O

OH

O

OH HOCH2

OH

O OH

OH

OH

OH

OH

O HO HO

XA3XA2+3XX DP9

O

HOCH2

O

HOCH2

HO

HO

HO

HO

O

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O

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HO

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O

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OH

OH

O

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O

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XU4m2XXA3XX DP9

O

HOCH2

HO

HO O

O HO

O

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O O

O O

OH

OH

OH

O O

OH

O

OH

OH

OH

O OH

OH

OH

O

O O

OH

OH

OH

H3CO OH

OH

Fig. S3. Nomenclature and structure of substituted xylan oligosaccharides described in this article. The nomenclature used is the one-letter code system proposed by Faure et al. (1), in which the side chains Araf (A) or (4-O-methyl-) glucuronic acid [U(4m)] replace the Xylp (X) of the backbone. Superscript numbers following the letter of a side chain indicate the linkage position. The degree of polymerization (DP) is given.

1. Faure R, et al. (2009) A brief and informationally rich naming system for oligosaccharide motifs of heteroxylans found in plant cell walls. Aust J Chem 62:533–537.

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A

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3xmyc

TaXAT2

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2

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68kDa

HSQC

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110.0

TOCSY

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13C

108.0

4.10 4.15

A3-Xmono

A3-Xdi A -Xdi 2

3 A2-Xdi A -Xdi

A3-Xmono

*

5.40

5.35 1H

5.30 5.25 (ppm)

5.20

Fig. S4. Expression of wheat TaXAT2-myc in Arabidopsis and NMR analyses of resulting xylan. (A) Overview of the pIRX9::TaXAT2-myc construct. The black box indicates the translated region with a 3× Myc tag translationally fused to the 3′ end of the ORF of TaXAT2. Light gray boxes (UTRs) and white boxes (intergenic regions) are genomic up- and downstream regions of IRX9 (At2g37090), arrow indicates the promoter. (B) Western blot analysis using a Mycantibody of three independent TaXAT2 transgenic lines in wild-type (1 and 3) and gux background (2). Molecular size of TaXAT2-Myc is 68 kDa. C, Control, nontransgenic Arabidopsis material. (C) NMR spectra of xylan oligosaccharides. Digested xylan derived from the 1 M KOH AIR fraction of Arabidopsis expressing TaXAT2 in a gux background (red) showing the presence of xylan monosubstituted with α-Araf (A3-Xmono); a peak at 5.39 ppm (marked with an asterisk) was identified and assigned as the H1 resonance of α-Araf (A3-Xmono). The assignment was confirmed by 2D TOCSY and 13C HSQC experiments that correlate the H1 with the H2 and C1 resonances, respectively, at their known positions. α-Araf substitution is absent in gux Arabidopsis xylan prepared by the same method (blue). Wheat arabinoxylan (black) is also shown for reference.

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Y6α

O

O

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B1β

OH HO

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OH HO

B4

Y6β 1218.0

954.2

C2

150

B3

C-2

E3

405

660

B

X4

1,5

B5

D5

G4

Y6α

X6

H1

915

Mass (m/z)

X6β Y6 1,4X Y6α 1,5 6α

E6

E5C5-2

G5

1,5

1137.0

822.2

B2

B4

B6

X5

1246.3 1276.0

322.3

231.2

OH

1,5

850.3 873.2

X1

Y3

G3

Y4

788.3

Y2

1,5

B1β

X3

690.3

271.3 294.3

B1α C1 B1

HO

B6

1,5

X2

1,5

O

H N

Y5

E2 Y1

HO

OH

O

OH

B5

OH

D

A

X1

O

O

OH

B3

Y6β

G3

O

O

Y1

1086.1 1121.1

H3CO

HO

HO

B2

COOH O

Y3 O X 2

X3

O

O

[M + Na]+

Y2 1,5

1,5

1005.1

OH HO

B1

Y4 1,5

X4

X5

O

HO

Y5 1,5

OH

982.2

X6

HO

HO

718.3

1170

1425 [M + Na]+

C

XU4m2XXA3XX-2-AA Y6 1,5

X6

1,5

O

HO

HO

O

OH HO

B1

O

HO

B2 O

COOH O

O

G3

O

OH HO

Y2 1,5

O

O

O

OH HO

B4

Y1

X1

2

O

OH

B3

Y6β

Y3 O1,5X

X3

O

D5

OH

Y4 1,5

X4

O

Y6α

HO

Y5 1,5

X5

O

HOH2C

1408.2

*

Y6 1,5

1,5

D5

B1α

1408.4

O

HOH2C

1304.0

XU4m2XXA3XX-2-AA

741.2

Β

+Arafase

609.2 625.3

*

OsXAT3 wt

524.3

+Arafase

551.3

*

OsXAT2 wt

426.3

+Arafase

477.3

TaXAT2 wt

454.3

A

B5

O

H N HO

OH

HO

OH

O

OH

B6

H3CO

OH

150

660

Mass (m/z)

915

1218.0

X6β

1,5

Y6α

1170

1304.0

1121.1

1,4 X6α Y6 1,5X 6 Y6α

1275.9

A 6 E6

3,5

1137.0

B5

B6

1049.1

954.1

5

X5

1,5

1005.0

822.2

850.3 873.1

A5

3,5

D5G

920.3

Y4

B4

718.3

609.2

Y3

653.2 690.2

477.2

524.3

426.3

405

W3

B3

X4

1,5

741.2

A4

3,5

Y2 B G3 2 454.3

322.2

271.2 294.2

Y1

E2

X2

1,5

Y6β

Y5

X3

1,5

X1

1,5

982.1

HO

H1 1425

Fig. S5. Characterization of the XAT-dependent arabinofuranosidase-sensitive xylan oligosaccharide derived from XAT transgenic plants in wildtype background. (A) PACE analysis of arabinofuranosidase-sensitivity of XAT-dependent xylan oligosaccharides after xylanase digest. Xylan fingerprint of transgenics expressing TaXAT2, OsXAT2, or OsXAT3 is shown in comparison with the arabinofuranosidase digested material (+Arafase). Boxed area shows six-times longer exposure of identical gel. Asterisks mark the oligosaccharide specific for the transgenic lines. Oligosaccharide assignment is as follows. A: X, B: XX, D: XU(4m)2XX. (B and C) Structural characterization of the XAT-dependent arabinofuranosidase-sensitive xylan oligosaccharide. NP-LC–MALDI-ToF/ToF-MS/MS of the Pent8MeHexA XU4m2XXA3XX labeled with 2-aminobenzoic acid (2-AA) derived from (B) TaXAT2 transgenics (m/z 1408.4 [M + Na]+) and (C) OsXAT3 transgenics (m/z 1408.2 [M + Na]+).

Anders et al. www.pnas.org/cgi/content/short/1115858109

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At3g18170 At3g18180 IRX3 GUX1 IRX9 IRX10 GUX2 IRX14

sperm cell peripheral endosperm micropyral endosperm endosperm node root xylem cell root cell replum xylem hypocotyl silique stem stem general seed coat testa (seed coat) pericycle hypocotyl stele root hair zone stamen inflorescence cell culture / primary cell seed leaf primordia flower petiole petal developing meristemoid (stomatal precursor) zone pedicel cotyledons shoot apex sepal shoot apical meristem juvenile leaf pistil cauline leaf seedling rosette root culture callus stigma radicle cork axillary bud shoot apex adult leaf senescent leaf axillary shoot imbibed seed root phloem cell epid. atrichoblasts endodermis + cortex lateral root cap endodermis root tip roots chalazal endosperm root cortex cell abscission zone chalazal seed coat meristematic zone suspensor elongation zone embryo ovule ovary guard cell protoplast protoplast lateral root pollen mesophyll cell protoplast

Fig. S6. Coexpression analysis of Arabidopsis GT61s. Hierarchical cluster of expression profiles of At3g18170 and At3g18180 along with profiles of known secondary cell wall genes including genes involved in glucuronoxylan (GX) biosynthesis using the Genevestigator tool (www.genevestigator.com).

Anders et al. www.pnas.org/cgi/content/short/1115858109

8 of 10

Table S1. Cell wall composition of wheat TaXAT1 RNAi (H) and azygous control (N) endosperm samples: Oligosaccharide abundances (HPAEC peak areas) Oligosaccharide Line

Transgene

Cadenza 1 P 2 P 3 P 4 P 5

H N value H N value H N value H N value H N value

P SED (on 28 df) LSD (5%) LSD (1%) Residual variance Technical variance

3

G3

G4

X

XX

XA XX

XA3A3XX

XA3XA3XX

XA2+3XX

XA3A2+3XX

XA3XA2+3XX

21.4 17.6 23.1 0.003 17.1 20.3 0.076 20.7 20.1 0.744 19.6 15.4 0.021 17.5 19.9 0.182 1.71 3.50 4.73 17.60 0.74

11.6 11.2 18.9