A Novel Rice Xylosyltransferase Catalyzes the Addition of 2-O-Xylosyl Side Chains onto the Xylan Backbone

Xylan is a major hemicellulose in both primary and secondary walls of grass species. It consists of a linear backbone of β-1,4-linked xylosyl residues that are often substituted with monosaccharides and disaccharides. Xylosyl substitutions directly on the xylan backbone have not been reported in grass species, and genes responsible for xylan substitutions in grass species have not been well elucidated. Here, we report functional characterization of a rice (Oryza sativa) GT61 glycosyltransferase, XYXT1 (xylan xylosyltransferase1), for its role in xylan substitutions. XYXT1 was found to be ubiquitously expressed in different rice organs and its encoded protein was targeted to the Golgi, the site for xylan biosynthesis. When expressed in the Arabidopsis gux1/2/3 triple mutant, in which xylan was completely devoid of sugar substitutions, XYXT1 was able to add xylosyl side chains onto xylan. Glycosyl linkage analysis and comprehensive structural characterization of xylooligomers generated by xylanase digestion of xylan from transgenic Arabidopsis plants expressing XYXT1 revealed that the side chain xylosyl residues were directly attached to the xylan backbone at O-2, a substituent not present in wild-type Arabidopsis xylan. XYXT1 was unable to add xylosyl residues onto the arabinosyl side chains of xylan when it was co-expressed with OsXAT2 (Oryza sativa xylan arabinosyltransferase2) in the gux1/2/3 triple mutant. Furthermore, we showed that recombinant XYXT1 possessed an activity transferring xylosyl side chains onto xylooligomer acceptors, whereas recombinant OsXAT2 catalyzed the addition of arabinosyl side chains onto xylooligomer acceptors. Our findings from both an in vivo gain-of-function study and an in vitro recombinant protein activity assay demonstrate that XYXT1 is a novel β-1,2-xylosyltransferase mediating the addition of xylosyl side chains onto xylan.