Abstract
Plant cell walls contain the hemicellulose xyloglucan, whose fine structure may vary depending on cell type, tissue, and/or plant species. Most but not all of the glycosyltransferases involved in the biosynthesis of xyloglucan sidechains have been identified. Here, we report the identification of several functional glycosyltransferases from blueberry (Vaccinium corymbosum bluecrop). Among those transferases is a hitherto elusive Xyloglucan:Beta-xylosylTransferase (XBT). Heterologous expression of VcXBT in the Arabidopsis thaliana double mutant mur3 xlt2, where xyloglucan consists only of an unsubstituted xylosylated glucan core structure, results in the production of the xylopyranose-containing "U" sidechain as characterized by mass spectrometry, glycosidic linkage, and NMR analysis. The introduction of the additional xylopyranosyl residue rescues the dwarfed phenotype of the untransformed Arabidopsis mur3 xlt2 mutant to wild-type height. Structural protein analysis using Alphafold of this and other related xyloglucan glycosyltransferase family 47 proteins not only identifies potential domains that might influence the regioselectivity of these enzymes but also gives hints to specific amino acids that might determine the donor-substrate specificity of these glycosyltransferases.