Functional analysis of GT61 glycosyltransferases from grass species in xylan substitutions

Main conclusion 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. Abstract Xylan is the major hemicellulose in the cell walls of grass species and it is typified by having arabinofuranosyl (Ara f ) 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 Ara f 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 Ara f 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.