Corrigendum: Modification of plant cell walls with hydroxycinnamic acids by BAHD acyltransferases.

[This corrects the article DOI: 10.3389/fpls.2022.1088879.].

Complex polyploid and hybrid species in an apomictic and sexual tropical forage grass group: genomic composition and evolution in Urochloa (Brachiaria) species.

BACKGROUND AND AIMS: Diploid and polyploid Urochloa (including Brachiaria, Panicum and Megathyrsus species) C4 tropical forage grasses originating from Africa are important for food security and the environment, often being planted in marginal lands worldwide. We aimed to characterize the nature of their genomes, the repetitive DNA and the genome composition of polyploids, leading to a model of the evolutionary pathways within the group including many apomictic species. METHODS: Some 362 forage grass accessions from international germplasm collections were studied, and ploidy was determined using an optimized flow cytometry method. Whole-genome survey sequencing and molecular cytogenetic analysis were used to identify chromosomes and genomes in Urochloa accessions belonging to the 'brizantha' and 'humidicola' agamic complexes and U. maxima. KEY RESULTS: Genome structures are complex and variable, with multiple ploidies and genome compositions within the species, and no clear geographical patterns. Sequence analysis of nine diploid and polyploid accessions enabled identification of abundant genome-specific repetitive DNA motifs. In situ hybridization with a combination of repetitive DNA and genomic DNA probes identified evolutionary divergence and allowed us to discriminate the different genomes present in polyploids. CONCLUSIONS: We suggest a new coherent nomenclature for the genomes present. We develop a model of evolution at the whole-genome level in diploid and polyploid accessions showing processes of grass evolution. We support the retention of narrow species concepts for Urochloa brizantha, U. decumbens and U. ruziziensis, and do not consider diploids and polyploids of single species as cytotypes. The results and model will be valuable in making rational choices of parents for new hybrids, assist in use of the germplasm for breeding and selection of Urochloa with improved sustainability and agronomic potential, and assist in measuring and conserving biodiversity in grasslands.

Modification of plant cell walls with hydroxycinnamic acids by BAHD acyltransferases.

In the last decade it has become clear that enzymes in the "BAHD" family of acyl-CoA transferases play important roles in the addition of phenolic acids to form ester-linked moieties on cell wall polymers. We focus here on the addition of two such phenolics-the hydroxycinnamates, ferulate and p-coumarate-to two cell wall polymers, glucuronoarabinoxylan and to lignin. The resulting ester-linked feruloyl and p-coumaroyl moities are key features of the cell walls of grasses and other commelinid monocots. The capacity of ferulate to participate in radical oxidative coupling means that its addition to glucuronoarabinoxylan or to lignin has profound implications for the properties of the cell wall - allowing respectively oxidative crosslinking to glucuronoarabinoxylan chains or introducing ester bonds into lignin polymers. A subclade of ~10 BAHD genes in grasses is now known to (1) contain genes strongly implicated in addition of p-coumarate or ferulate to glucuronoarabinoxylan (2) encode enzymes that add p-coumarate or ferulate to lignin precursors. Here, we review the evidence for functions of these genes and the biotechnological applications of manipulating them, discuss our understanding of mechanisms involved, and highlight outstanding questions for future research.

RNAi suppression of xylan synthase genes in wheat starchy endosperm.

The xylan backbone of arabinoxylan (AX), the major cell wall polysaccharide in the wheat starchy endosperm, is synthesised by xylan synthase which is a complex of three subunits encoded by the GT43_1, GT43_2 and GT47_2 genes. RNAi knock-down of either GT43_1 or all three genes (triple lines) resulted in decreased AX measured by digestion with endoxylanase (to 33 and 34.9% of the controls) and by monosaccharide analysis (to 45.9% and 47.4% of the controls) with greater effects on the amount of water-extractable AX (to 20.6 and 19.9% of the controls). Both sets of RNAi lines also had greater decreases in the amounts of substituted oligosaccharides released by digestion of AX with endoxylanase than in fragments derived only from the xylan backbone. Although the GT43_1 and triple lines had similar effects on AX they did differ in their contents of soluble sugars (increased in triple only) and on grain size (decreased in triple only). Both sets of transgenic lines had decreased grain hardness, indicating effects on cell wall mechanics. These results, and previously published studies of RNAi suppression of GT43_2 and GT47_2 and of a triple mutant of GT43_2, are consistent with the model of xylan synthase comprising three subunits one of which (GT47_2) is responsible for catalysis with the other two subunits being required for correct functioning but indicate that separate xylan synthase complexes may be responsible for the synthesis of populations of AX which differ in their structure and solubility.

Flow Cytometry-Based Determination of Ploidy from Dried Leaf Specimens in Genomically Complex Collections of the Tropical Forage Grass Urochloa s. l.

Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We aimed to develop an optimized approach to determine ploidy of germplasm collection of this tropical forage grass group using dried leaf material, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Our methods enable robust identification of ploidy levels (coefficient of variation of G0/G1 peaks, CV, typically <5%). Ploidy of some 348 forage grass accessions (ploidy range from 2x to 9x), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes are used in the current breeding programs at CIAT and EMBRAPA: the 'brizantha' and 'humidicola' agamic complexes are variable, with multiple ploidy levels. Some U. brizantha accessions have odd level of ploidy (5x), and the relative differences in fluorescence values of the peak positions between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed aneuploidy.

Allele mining in diverse accessions of tropical grasses to improve forage quality and reduce environmental impact.

BACKGROUND AND AIMS: The C4Urochloa species (syn. Brachiaria) and Megathyrsus maximus (syn. Panicum maximum) are used as pasture for cattle across vast areas in tropical agriculture systems in Africa and South America. A key target for variety improvement is forage quality: enhanced digestibility could decrease the amount of land required per unit production, and enhanced lipid content could decrease methane emissions from cattle. For these traits, loss-of-function (LOF) alleles in known gene targets are predicted to improve them, making a reverse genetics approach of allele mining feasible. We therefore set out to look for such alleles in diverse accessions of Urochloa species and Megathyrsus maximus from the genebank collection held at the CIAT. METHODS: We studied allelic diversity of 20 target genes (11 for digestibility, nine for lipid content) in 104 accessions selected to represent genetic diversity and ploidy levels of U. brizantha, U. decumbens, U. humidicola, U. ruziziensis and M. maximum. We used RNA sequencing and then bait capture DNA sequencing to improve gene models in a U. ruziziensis reference genome to assign polymorphisms with high confidence. KEY RESULTS: We found 953 non-synonymous polymorphisms across all genes and accessions; within these, we identified seven putative LOF alleles with high confidence, including those in the non-redundant SDP1 and BAHD01 genes present in diploid and tetraploid accessions. These LOF alleles could respectively confer increased lipid content and digestibility if incorporated into a breeding programme. CONCLUSIONS: We demonstrated a novel, effective approach to allele discovery in diverse accessions using a draft reference genome from a single species. We used this to find gene variants in a collection of tropical grasses that could help reduce the environmental impact of cattle production.

Accumulation and deposition of triacylglycerols in the starchy endosperm of wheat grain.

A combination of lipidomics, transcriptomics and bioimaging has been used to study triacylglycerol synthesis and deposition in the developing starchy endosperm of wheat. The content of TAG increased between 14 and 34 days after anthesis, from 50 to 115 mg/100 g dry wt and from about 35 to 175 mg/100 g dry wt in two experiments. The major fatty acids were C16 (palmitic C16:0 and palmitoleic C16:1) and C18 (stearic C18:0, oleic C18:1, linoleic C18:2 and linolenic C18:3), with unsaturated fatty acids accounting for about 75-80% of the total throughout development. Linoleic acid (C18:2) was the major component at all stages and the proportion increased during development. Transcript profiling indicated that predominant route to TAG synthesis and oil accumulation is via the Kennedy pathway and diacylglycerol acyltransferase (DGAT) activity. Confocal microscopy of stained tissue sections showed that TAG accumulated in droplets which are associated with protein and concentrated in the starchy endosperm cells below the sub-aleurone cells. Transcripts encoding 16kd oleosins were also expressed, indicating that the oil droplets are in part stabilised by oleosin proteins.

The TaCslA12 gene expressed in the wheat grain endosperm synthesizes wheat-like mannan when expressed in yeast and Arabidopsis.

Mannan is a class of cell wall polysaccharides widespread in the plant kingdom. Mannan structure and properties vary according to species and organ. The cell walls of cereal grains have been extensively studied due to their role in cereal processing and to their beneficial effect on human health as dietary fiber. Recently, we showed that mannan in wheat (Triticum aestivum) grain endosperm has a linear structure of ß-1,4-linked mannose residues. The aim of this work was to study the biosynthesis and function of wheat grain mannan. We showed that mannan is deposited in the endosperm early during grain development, and we identified candidate mannan biosynthetic genes expressed in the endosperm. The functional study in wheat was unsuccessful therefore our best candidate genes were expressed in heterologous systems. The endosperm-specificTaCslA12 gene expressed in Pichia pastoris and in an Arabidopsis thaliana mutant depleted in glucomannan led to the production of wheat-like linear mannan lacking glucose residues and with moderate acetylation. Therefore, this gene encodes a mannan synthase and is likely responsible for the synthesis of wheat endosperm mannan.

Suppression of a BAHD acyltransferase decreases p-coumaroyl on arabinoxylan and improves biomass digestibility in the model grass Setaria viridis.

Grass cell walls have hydroxycinnamic acids attached to arabinosyl residues of arabinoxylan (AX), and certain BAHD acyltransferases are involved in their addition. In this study, we characterized one of these BAHD genes in the cell wall of the model grass Setaria viridis. RNAi silenced lines of S. viridis (SvBAHD05) presented a decrease of up to 42% of ester-linked p-coumarate (pCA) and 50% of pCA-arabinofuranosyl, across three generations. Biomass from SvBAHD05 silenced plants exhibited up to 32% increase in biomass saccharification after acid pre-treatment, with no change in total lignin. Molecular dynamics simulations suggested that SvBAHD05 is a p-coumaroyl coenzyme A transferase (PAT) mainly involved in the addition of pCA to the arabinofuranosyl residues of AX in Setaria. Thus, our results provide evidence of p-coumaroylation of AX promoted by SvBAHD05 acyltransferase in the cell wall of the model grass S. viridis. Furthermore, SvBAHD05 is a promising biotechnological target to engineer crops for improved biomass digestibility for biofuels, biorefineries and animal feeding.

Loss of TaIRX9b gene function in wheat decreases chain length and amount of arabinoxylan in grain but increases cross-linking.

Wheat contains abundant xylan in cell walls of all tissues, but in endosperm, there is an unusual form of xylan substituted only by arabinose (arabinoxylan; AX) that has long chains and low levels of feruloylation, a fraction of which is extractable in water (WE-AX). WE-AX acts as soluble dietary fibre but also gives rise to viscous extracts from grain, a detrimental trait for some non-food uses of wheat. Here, we show that a glycosyl transferase family 43 wheat gene abundantly expressed in endosperm complements the Arabidopsis irx9 mutant and so name the three homoeologous genes TaIRX9b. We generated wheat lines with a constitutive knockout of TaIRX9b by stacking loss-of-function alleles for these homeologues from a mutagenized hexaploid wheat population resulting in decreases in grain extract viscosity of 50%-80%. The amount and chain length of WE-AX molecules from grain of these triple-stack lines was decreased accounting for the changes in extract viscosity. Imaging of immature wheat grain sections of triple-stacks showed abolition of immunolabelling in endosperm with LM11 antibody that recognizes epitopes in AX, but also showed apparently normal cell size and shape in all cell types, including endosperm. We identified differentially expressed genes from endosperm of triple-stacks suggesting that compensatory changes occur to maintain this endosperm cell wall integrity. Consistent with this, we observed increased ferulate dimerization and increased cross-linking of WE-AX molecules in triple-stacks. These novel wheat lines lacking functional TaIRX9b therefore provide insight into control of wheat endosperm cell walls.

High post-anthesis temperature effects on bread wheat (Triticum aestivum L.) grain transcriptome during early grain-filling.

BACKGROUND: High post-anthesis (p.a) temperatures reduce mature grain weights in wheat and other cereals. However, the causes of this reduction are not entirely known. Control of grain expansion by the maternally derived pericarp of the grain has previously been suggested, although this interaction has not been investigated under high p.a. temperatures. Down-regulation of pericarp localised genes that regulate cell wall expansion under high p.a. temperatures may limit expansion of the encapsulated endosperm due to a loss of plasticity in the pericarp, reducing mature grain weight. Here the effect of high p.a. temperatures on the transcriptome of the pericarp and endosperm of the wheat grain during early grain-filling was investigated via RNA-Seq and is discussed alongside grain moisture dynamics during early grain development and mature grain weight. RESULTS: High p.a. temperatures applied from 6-days after anthesis (daa) and until 18daa reduced the grain's ability to accumulate water, with total grain moisture and percentage grain moisture content being significantly reduced from 14daa onwards. Mature grain weight was also significantly reduced by the same high p.a. temperatures applied from 6daa for 4-days or more, in a separate experiment. Comparison of our RNA-Seq data from whole grains, with existing data sets from isolated pericarp and endosperm tissues enabled the identification of subsets of genes whose expression was significantly affected by high p.a. temperature and predominantly expressed in either tissue. Hierarchical clustering and gene ontology analysis resulted in the identification of a number of genes implicated in the regulation of cell wall expansion, predominantly expressed in the pericarp and significantly down-regulated under high p.a. temperatures, including endoglucanase, xyloglucan endotransglycosylases and a ß-expansin. An over-representation of genes involved in the 'cuticle development' functional pathway that were expressed in the pericarp and affected by high p.a. temperatures was also observed. CONCLUSIONS: High p.a. temperature induced down-regulation of genes involved in regulating pericarp cell wall expansion. This concomitant down-regulation with a reduction in total grain moisture content and grain weight following the same treatment period, adds support to the theory that high p.a. temperatures may cause a reduction in mature grain weight as result of decreased pericarp cell wall expansion.

Identification of a major QTL and associated molecular marker for high arabinoxylan fibre in white wheat flour.

Dietary fibre (DF) has multiple health benefits and wheat grains are major sources of DF for human health. However, DF is depleted in white wheat flour which is more widely consumed than wholegrain. The major DF component in white flour is the cell wall polysaccharide arabinoxylan (AX). We have identified the Chinese wheat cultivar Yumai 34 as having unusually high contents of AX in both water-soluble and insoluble forms. We have therefore used populations generated from crosses between Yumai 34 and four other wheat cultivars, three with average contents of AX (Ukrainka, Altigo and Claire) and one also having unusually high AX (Valoris), in order to map QTLs for soluble AX (determined as relative viscosity of aqueous extracts of wholemeal flours) and total AX (determined by enzyme fingerprinting of white flour). A number of QTL were mapped, but most were only detected in one or two crosses. However, all four crosses showed strong QTLs for high RV/total AX on chromosome 1B, with Yumai 34 being the increasing parent, and a KASP marker for the Yumai 34 high AX allele was validated by analysis of high AX lines derived from Yumai 34 but selected by biochemical analysis. A QTL for RV was also mapped on chromosome 6B in Yumai 34 x Valoris, with Valoris being the increasing allele, which is consistent with the observation of transgressive segregation for this population. Association studies in an independent germplasm panel identified marker trait associations for relative viscosity in these same locations while direct selection for fibre content in breeding resulted in high levels of enrichment for the Yumai 34 1B allele. The data therefore indicate that marker-assisted breeding can be used to develop wheat with high AX fibre in white flour.

Response of cell-wall composition and RNA-seq transcriptome to methyl-jasmonate in Brachypodium distachyon callus.

MAIN CONCLUSION: Methyl-jasmonate induces large increases in p-coumarate linked to arabinoxylan in Brachypodium and in abundance of GT61 and BAHD family transcripts consistent with a role in synthesis of this linkage. Jasmonic acid (JA) signalling is required for many stress responses in plants, inducing large changes in the transcriptome, including up-regulation of transcripts associated with lignification. However, less is known about the response to JA of grass cell walls and the monocot-specific features of arabinoxylan (AX) synthesis and acylation by ferulic acid (FA) and para-coumaric acid (pCA). Here, we show that methyl-jasmonate (MeJA) induces moderate increases in FA monomer, > 50% increases in FA dimers, and five-sixfold increases in pCA ester-linked to cell walls in Brachypodium callus. Direct measurement of arabinose acylated by pCA (Araf-pCA) indicated that most or all the increase in cell-wall pCA was due to pCA ester-linked to AX. Analysis of the RNA-seq transcriptome of the callus response showed that these cell-wall changes were accompanied by up-regulation of members of the GT61 and BAHD gene families implicated in AX decoration and acylation; two BAHD paralogues were among the most up-regulated cell-wall genes (seven and fivefold) after 24 h exposure to MeJA. Similar responses to JA of orthologous BAHD and GT61 transcripts are present in the RiceXPro public expression data set for rice seedlings, showing that they are not specific to Brachypodium or to callus. The large response of AX-pCA to MeJA may, therefore, indicate an important role for this linkage in response of primary cell walls of grasses to JA signalling.

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

Spatiotemporal expression patterns of wheat amino acid transporters reveal their putative roles in nitrogen transport and responses to abiotic stress.

Amino acid transporters have roles in amino acid uptake from soil, long-distance transport, remobilization from vegetative tissues and accumulation in grain. Critically, the majority of wheat grain nitrogen is derived from amino acids remobilized from vegetative organs. However, no systematic analysis of wheat AAT genes has been reported to date. Here, 283 full length wheat AAT genes representing 100 distinct groups of homeologs were identified and curated by selectively consolidating IWGSC CSSv2 and TGACv1 Triticum aestivum genome assemblies and reassembling or mapping of IWGSC CSS chromosome sorted reads to fill any gaps. Gene expression profiling was performed using public RNA-seq data from root, leaf, stem, spike, grain and grain cells (transfer cell (TC), aleurone cell (AL), and starchy endosperm (SE)). AATs highly expressed in roots are good candidates for amino acid uptake from soil whilst AATs highly expressed in senescing leaves and stems may be involved in translocation to grain. AATs in TC (TaAAP2 and TaAAP19) and SE (TaAAP13) may play important roles in determining grain protein content and grain yield. The expression levels of AAT homeologs showed unequal contributions in response to abiotic stresses and development, which may aid wheat adaptation to a wide range of environments.

Feruloylation and structure of arabinoxylan in wheat endosperm cell walls from RNAi lines with suppression of genes responsible for backbone synthesis and decoration.

Arabinoxylan (AX) is the major component of the cell walls of wheat grain (70% in starchy endosperm), is an important determinant of end-use qualities affecting food processing, use for animal feed and distilling and is a major source of dietary fibre in the human diet. AX is a heterogeneous polysaccharide composed of fractions which can be sequentially extracted by water (WE-AX), then xylanase action (XE-AX) leaving an unextractable (XU-AX) fraction. We determined arabinosylation and feruloylation of AX in these fractions in both wild-type wheat and RNAi lines with decreased AX content (TaGT43_2 RNAi, TaGT47_2 RNAi) or decreased arabinose 3-linked to mono-substituted xylose (TaXAT1 RNAi). We show that these fractions are characterized by the degree of feruloylation of AX, <5, 5-7 and 13-19 mg bound ferulate (g-1 AX), and their content of diferulates (diFA), <0.3, 1-1.7 and 4-5 mg (g-1 AX), for the WE, XE and XU fractions, respectively, in all RNAi lines and their control lines. The amount of AX and its degree of arabinosylation and feruloylation were less affected by RNAi transgenes in the XE-AX fraction than in the WE-AX fraction and largely unaffected in the XU-AX fraction. As the majority of diFA is associated with the XU-AX fraction, there was only a small effect (TaGT43_2 RNAi, TaGT47_2 RNAi) or no effect (TaXAT1 RNAi) on total diFA content. Our results are compatible with a model where, to maintain cell wall function, diFA is maintained at stable levels when other AX properties are altered.

G-fibre cell wall development in willow stems during tension wood induction.

Willows (Salix spp.) are important as a potential feedstock for bioenergy and biofuels. Previous work suggested that reaction wood (RW) formation could be a desirable trait for biofuel production in willows as it is associated with increased glucose yields, but willow RW has not been characterized for cell wall components. Fasciclin-like arabinogalactan (FLA) proteins are highly up-regulated in RW of poplars and are considered to be involved in cell adhesion and cellulose biosynthesis. COBRA genes are involved in anisotropic cell expansion by modulating the orientation of cellulose microfibril deposition. This study determined the temporal and spatial deposition of non-cellulosic polysaccharides in cell walls of the tension wood (TW) component of willow RW and compared it with opposite wood (OW) and normal wood (NW) using specific antibodies and confocal laser scanning microscopy and transmission electron microscopy. In addition, the expression patterns of an FLA gene (SxFLA12) and a COBRA-like gene (SxCOBL4) were compared using RNA in situ hybridization. Deposition of the non-cellulosic polysaccharides (1-4)-ß-D-galactan, mannan and de-esterified homogalacturonan was found to be highly associated with TW, often with the G-layer itself. Of particular interest was that the G-layer itself can be highly enriched in (1-4)-ß-D-galactan, especially in G-fibres where the G-layer is still thickening, which contrasts with previous studies in poplar. Only xylan showed a similar distribution in TW, OW, and NW, being restricted to the secondary cell wall layers. SxFLA12 and SxCOBL4 transcripts were specifically expressed in developing TW, confirming their importance. A model of polysaccharides distribution in developing willow G-fibre cells is presented.

Secondary cell wall composition and candidate gene expression in developing willow (Salix purpurea) stems.

The properties of the secondary cell wall (SCW) in willow largely determine the suitability of willow biomass feedstock for potential bioenergy and biofuel applications. SCW development has been little studied in willow and it is not known how willow compares with model species, particularly the closely related genus Populus. To address this and relate SCW synthesis to candidate genes in willow, a tractable bud culture-derived system was developed in Salix purpurea, and cell wall composition and RNA-Seq transcriptome were followed in stems during early development. A large increase in SCW deposition in the period 0-2 weeks after transfer to soil was characterised by a big increase in xylan content, but no change in the frequency of substitution of xylan with glucuronic acid, and increased abundance of putative transcripts for synthesis of SCW cellulose, xylan and lignin. Histochemical staining and immunolabeling revealed that increased deposition of lignin and xylan was associated with xylem, xylem fibre cells and phloem fibre cells. Transcripts orthologous to those encoding xylan synthase components IRX9 and IRX10 and xylan glucuronyl transferase GUX1 in Arabidopsis were co-expressed, and showed the same spatial pattern of expression revealed by in situ hybridisation at four developmental stages, with abundant expression in proto-xylem, xylem fibre and ray parenchyma cells and some expression in phloem fibre cells. The results show a close similarity with SCW development in Populus species, but also give novel information on the relationship between spatial and temporal variation in xylan-related transcripts and xylan composition.

RNA interference suppression of genes in glycosyl transferase families 43 and 47 in wheat starchy endosperm causes large decreases in arabinoxylan content.

The cell walls of wheat (Triticum aestivum) starchy endosperm are dominated by arabinoxylan (AX), accounting for 65% to 70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRREGULAR XYLEM9 [IRX9] and IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologs of these have been implicated in the synthesis of xylan in other species. Here, homologs of IRX10 TaGT47_2 and IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNA interference (RNAi) constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40% to 50% and the degree of arabinosylation was increased by 25% to 30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabeling for xylan and arabinoxylan epitopes and approximately 50% decreased cell wall thickness compared with controls. The proportion of AX that was water soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, the long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fiber in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.