Functional analysis of GT61 glycosyltransferases from grass species in xylan substitutions

Multiple rice GT61 members were demonstrated to be xylan arabinosyltransferases (XATs) mediating 3-O-arabinosylation of xylan and the functions of XATs and xylan 2-O-xylosyltransferases were shown to be conserved in grass species. Xylan is the major hemicellulose in the cell walls of grass species and it is typified by having arabinofuranosyl (Araf) substitutions. In this report, we demonstrated that four previously uncharacterized, Golgi-localized glycosyltransferases residing in clade A or B of the rice GT61 family were able to mediate 3-O-arabinosylation of xylan when heterologously expressed in the Arabidopsis gux1/2/3 triple mutant. Biochemical characterization of their recombinant proteins established that they were xylan arabinosyltransferases (XATs) capable of transferring Araf residues onto xylohexaose acceptors, and thus they were named OsXAT4, OsXAT5, OsXAT6 and OsXAT7. OsXAT5 and the previously identified OsXAT2 were shown to be able to arabinosylate xylooligomers with a degree of polymerization of as low as 3. Furthermore, a number of XAT homologs from maize, sorghum, Brachypodium and switchgrass were found to exhibit activities catalyzing Araf transfer onto xylohexaose, indicating that they are XATs involved in xylan arabinosylation in these grass species. Moreover, we revealed that homologs of another GT61 member, xylan 2-O-xylosyltransferase (XYXT1), from these grass species could mediate 2-O-xylosylation of xylan when expressed in the Arabidopsis gux1/2/3 mutant. Together, our findings indicate that multiple OsXATs are involved in 3-O-arabinosylation of xylan and the functions of XATs and XYXTs are conserved in grass species.