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

Abstract

Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with a-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Consistent with its function as a xylan arabinosyl 2-Oxylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.

An update on xylan structure, biosynthesis, and potential commercial applications

“…The enzymes included in this figure have been functionally characterized in vitro and the CAZy family is indicated for glycosyltransferases (GTs). The following references provide supporting data for the proteins listed herein: XYS/IRX10 ( Jensen et al, 2014 , Urbanowicz et al, 2014 ); GUX ( Lee et al, 2012 , Rennie et al, 2012 ); XAT/XYXT ( Anders et al, 2012 , Zhong et al, 2022b ); XAXT ( Zhong et al, 2022a ); XYXT ( Zhong et al, 2018 ); GXMT ( Urbanowicz et al, 2012 ); XOAT/TBL ( Lunin et al, 2020 , Urbanowicz et al, 2014 ); and X3AT1/X2AT1 ( Zhong et al, 2022b ). Red asterisks (*) indicate the linkage generated by the action of the respective enzyme.…”

Altering the substitution and cross‐linking of glucuronoarabinoxylans affects cell wall architecture in Brachypodium distachyon

“…We recently proposed that Os XAX1 functions as a glycosyltransferase adding hydroxycinnamic acid‐modified Ara f substitutions to xylan since xylan osxax1 mutant exhibits a reduction in both hydroxycinnamate‐modified Ara f monosaccharide and Xyl p ‐Ara f disaccharide substitutions (Feijao et al ., 2022). This hypothesis was supported by the identification of another member of the GT61 family that acts as a xylan Ara f 2‐ O ‐xylosyl‐transferase to generate the Xyl p ‐Ara f disaccharide substitutions (Zhong et al ., 2022). Rice xax1 exhibited changes in cell wall cross‐linking, demonstrated by reduction in diverse hydroxycinnamate–hydroxycinnamate and monolignol–hydroxycinnamate coupling products (Feijao et al ., 2022).…”

“…Although the substitutions consist mainly of Ara f and GlcA similar to other plants (Tryfona et al ., 2019), there are additionally, hydroxycinnamic acid groups (e.g. feruloyl (Fer)‐ or coumaryl (Co) group) esterified to the O ‐5 position of Ara f residues (Mueller‐Harvey et al ., 1986; Hatfield et al ., 2017; Feijao et al ., 2022), and also Xyl p ‐Ara f substitutions (Wende & Fry, 1997; Tryfona et al ., 2019; Zhong et al ., 2022). Ferulation of grass xylan facilitates its dimerization via diferulate bridge formation or cross‐linking to lignin with coniferyl or sinapyl lignin units (Hatfield et al ., 1999, 2017), possibly increasing the strength and recalcitrance of the wall (Buanafina et al ., 2010; Ralph, 2010; Vogel et al ., 2010; de Souza et al ., 2018).…”

Grass xylan structural variation suggests functional specialization and distinctive interaction with cellulose and lignin

“…Grass xylan substitution consists mainly of Ara f , with comparatively fewer GlcA substitutions (Ebringerová & Heinze, 2000 ; Tryfona et al., 2019 ). However, grass xylans have some unique structural features, such as Xyl p –Ara f substitutions (Ebringerová & Heinze, 2000 ; Tryfona et al., 2019 ; Wende & Fry, 1997 ; Zhong et al., 2022 ) or hydroxycinnamic acid groups, for example, feruloyl (Fer) or coumaryl (Co) groups, esterified to the O ‐5 position of Ara f residues (Feijao et al., 2021 ; Hatfield et al., 2017 ; Mueller‐Harvey et al., 1986 ). These hydroxycinnamic groups can be esterified or etherified to lignin (Hatfield et al., 2017 ).…”