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

Abstract

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.type II cell walls | second-generation biofuels | dietary fiber C 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 gl...

Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos

“…Furthermore, substitutions of HX backbone with arabinosyl residues is catalyzed by GT61 family enzymes (Anders, Wilkinson, 2012, Chiniquy et al , 2012. A decrease in transcription activities of GT61 family genes (MLOC_68728 and MLOC_6357, Fig.2c) is as expected, since the ratios of substituted /unsubstituted HX decrease in germinating embryos (Table 1).…”

“…HX backbones are synthesized by members of the glycosyltransferase family 43 (GT43) and glycosyltransferase family 47 (GT47) (Scheller and Ulvskov, 2010). The glucuronosyl substitution of the backbone is catalyzed by members of the glycosyltransferase family 8 (GT8) (Mortimer et al , 2010), while the -(1,3)-linked arabinosyl substitution for the HX backbone is catalyzed by members of the glycosyltransferase family 61 (GT61) (Anders et al , 2012). Members of the cellulose synthase-like family F (CslF) and cellulose synthase-like family H (CslH) (both GT2) are responsible for the biosynthesis of (1,3)(1,4)-β-D-glucans (Burton et al , 2006, Doblin et al , 2009).…”

RNA Interference Suppression of Genes in Glycosyl Transferase Families 43 and 47 in Wheat Starchy Endosperm Causes Large Decreases in Arabinoxylan Content

“…In Arabidopsis, xylan GlcA transferase proteins from GT8 have been shown to confer this activity (Mortimer et al, 2010). Also, GT61 proteins have been shown to be xylan arabinosyl transferases in grasses, including wheat (Anders et al, 2012). While only a IRX14 homolog was detected in this complex, of great interest is whether a IRX9 homolog is also required in grasses for xylan backbone extension, as appears to be the case in woody and herbaceous dicots (Brown et al, 2007;Pena et al, 2007;Lee et al, 2011Lee et al, , 2012.…”

“…Using a strong starchy endosperm-specific promoter to drive RNA interference (RNAi) to suppress specific transcripts, we have demonstrated the key role of TaCSLF6 in synthesizing 1,3;1,4-b-D-glucan (Nemeth et al, 2010) and that the GT61 gene TaXAT1 is responsible for nearly all the monosubstituted Ara in AX in endosperm (Anders et al, 2012). The amount and structure of AX in wheat starchy endosperm are also of great practical importance; as it is the main component of dietary fiber in wheat foods, it is a major contributor of dietary fiber in the human diet (Topping, 2007), and it also affects the processing properties of wheat flour for different end uses (Saulnier et al, 2007).…”

The Role of the Plant-Specific ALTERED XYLOGLUCAN9 Protein in Arabidopsis Cell Wall PolysaccharideO-Acetylation

“…While mutants defective for cellulose synthase and xylan backbone biosynthesis have obvious implications for wall integrity, the connection between wall integrity and xylan O-acetylation is less clear. Mutants lacking xylan side chains such as glucuronosyl (Mortimer et al, 2010), arabinosyl (Anders et al, 2012), or xylosyl (Chiniquy et al, 2012) residues have not been reported to have such a strong phenotype. This indicates that the acetyl substituent is unique in having such a large impact on the functional properties of xylan.…”