Hydroxycinnamic acid‐modified xylan side chains and their cross‐linking products in rice cell walls are reduced in the Xylosyl arabinosyl substitution of xylan 1 mutant

Abstract

The intricate architecture of cell walls and the complex cross-linking of their components hinders some industrial and agricultural applications of plant biomass. Xylan is a key structural element of grass cell walls, closely interacting with other cell wall components such as cellulose and lignin. The main branching points of grass xylan, 3-linked L-arabinosyl substitutions, can be modified by ferulic acid (a hydroxycinnamic acid), which cross-links xylan to other xylan chains and lignin. XAX1 (Xylosyl arabinosyl substitution of xylan 1), a rice (Oryza sativa) member of the glycosyltransferase family GT61, has been described to add xylosyl residues to arabinosyl substitutions modified by ferulic acid. In this study, we characterize hydroxycinnamic acid-decorated arabinosyl substitutions present on rice xylan and their cross-linking, in order to decipher the role of XAX1 in xylan synthesis. Our results show a general reduction of hydroxycinnamic acidmodified 3-linked arabinosyl substitutions in xax1 mutant rice regardless of their modification with a xylosyl residue. Moreover, structures resembling the direct cross-link between xylan and lignin (ferulated arabinosyl substitutions bound to lignin monomers and dimers), together with diferulates known to cross-link xylan, are strongly reduced in xax1. Interestingly, apart from feruloyl and p-coumaroyl modifications on arabinose, putative caffeoyl and oxalyl modifications were characterized, which were also reduced in xax1. Our results suggest an alternative function of XAX1 in the transfer of hydroxycinnamic acid-modified arabinosyl substitutions to xylan, rather than xylosyl transfer to arabinosyl substitutions. Ultimately, XAX1 plays a fundamental role in cross-linking, providing a potential target for the improvement of use of grass biomass.

Identification of xylan arabinosyl 2‐O‐xylosyltransferases catalyzing the addition of 2‐O‐xylosyl residue onto arabinosyl side chains of xylan in grass species

“…Previous studies of rice GT61 members showed that eight of them, including OsXAT2–OsXAT7, OsXYXT1, and OsXAX1, mediate 3‐ O ‐Ara f , 2‐O‐ Xyl, and hydroxycinnamate‐esterified 3‐ O ‐Ara f substitutions of xylan, respectively (Anders et al., 2012; Chiniquy et al., 2012; Zhong, Cui, Phillips, & Ye, 2018; Zhong et al., 2021; Feijao et al., 2022). Whether any of the remaining rice GT61 members are involved in xylan substitutions remains elusive.…”

“…Grass xylan is heavily substituted with 3‐ O ‐Ara f side chains, some of which can be further decorated at O ‐2 with Xyl or Ara f . Although an early study suggested that rice XAX1 was likely responsible for Xyl transfer onto the Ara f residues of xylan to generate the Xyl‐Ara f side chains (Chiniquy et al., 2012), a recent report proposes that XAX1 functions to transfer hydroxycinnamate‐esterified Ara f residues onto xylan based on the finding that the xax1 mutant has a general reduction of hydroxycinnamate‐esterified Ara f residues in xylan regardless of whether Ara f is substituted with Xyl or not (Feijao et al., 2022). Therefore, glycosyltransferases involved in the transfer of Xyl or Ara f onto the 3‐ O ‐Ara f residues of xylan to form the Xyl‐Ara f and Ara f ‐Ara f disaccharide side chains remain elusive (Zhong et al., 2019).…”

“…Based on the observed mutant chemotypes, it was proposed that XAX1 might be a xylosyltransferase adding Xyl onto the Ara f residues of xylan to form the Xyl‐Ara f side chains and hydroxycinnamate esterification of Ara f might occur after Xyl is added onto the Ara f residues (Chiniquy et al., 2012). However, a recent report showed that rather than a specific reduction of hydroxycinnamate esterification of Ara f residues in the Xyl‐Ara f side chains, the xax1 mutation caused a general reduction of hydroxycinnamate‐esterified Ara f in both Ara f monosaccharide and Xyl‐Ara f disaccharide substituents (Feijao et al., 2022). This finding was explained by the suggestion that XAX1 may function as a glycosyltransferase adding hydroxycinnamate‐esterified Ara f directly onto the xylan backbone and another unknown xylosyltransferase subsequently transfers a Xyl residue onto the hydroxycinnamate‐esterified Ara f side chains of xylan (Feijao et al., 2022).…”

“…However, a recent report showed that rather than a specific reduction of hydroxycinnamate esterification of Ara f residues in the Xyl‐Ara f side chains, the xax1 mutation caused a general reduction of hydroxycinnamate‐esterified Ara f in both Ara f monosaccharide and Xyl‐Ara f disaccharide substituents (Feijao et al., 2022). This finding was explained by the suggestion that XAX1 may function as a glycosyltransferase adding hydroxycinnamate‐esterified Ara f directly onto the xylan backbone and another unknown xylosyltransferase subsequently transfers a Xyl residue onto the hydroxycinnamate‐esterified Ara f side chains of xylan (Feijao et al., 2022). This proposition seems to be congruent with the proposed functions of the cytosolic BAHD acyltransferases in generating UDP‐Ara f ‐hydroxycinnamate that is presumably transported into the Golgi and used as a donor by XAX1.…”

2D-HSQC-NMR-Based Screening of Feruloylated Side-Chains of Cereal Grain Arabinoxylans

“…A study focused on the maize grain fraction resistant to mild acid pretreatment and subsequent enzymatic saccharification found that feruloylated oligosaccharides made up 39% of the enzyme-resistant oligosaccharides, and most of these feruloylated oligosaccharides contained either the complex, feruloylated trisaccharide side-chain FAXG (α- l -galactopyranosyl-(1 → 2)-β- d -xylopyranosyl-(1 → 2)-5- O - trans -feruloyl- l -arabinofuranose; see Figure 1 ) or the feruloylated disaccharide FAX (β- d -xylopyranosyl-(1 → 2)-5- O - trans -feruloyl- l -arabinofuranose; see Figure 1 ) side-chains ( Appeldoorn et al, 2013 ). Because more complex AX structures contain additional glycosidic linkage types compared to simple AX ( Feijao et al, 2022 ), more elaborate enzymatic machinery is needed to break down these complex AX ( Rogowski et al, 2015 ; Beri et al, 2020 ). Unique microbial growth and fermentation patterns thus arise from different AX based on which microbial population is best equipped to compete for energy from the AX food source, i.e., produce the enzymes needed to break down the polymer into fermentable monosaccharides ( Yang et al, 2014 ; Centanni et al, 2017 ; Mendis et al, 2018 ; Ndeh and Gilbert, 2018 ).…”