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.

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) ¹H-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-X_mono), α-(1,3)–linked (A³-X_di), and α-(1,2)–linked (A²-X_di) to di-substituted Xylp and for Araf in arabinogalactan peptide (A-AGP).

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 five-times 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.

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 NP-HPLC-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. 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.