Biochemical characterization of family 43 glycosyltransferases in the Populus xylem: challenges and prospects

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Wood formation is a biological process of great economical importance. Genes active during the secondary cell wall formation of wood fibers from Populus tremula×tremuloides were previously identified by expression profiling through microarray analyses. A number of these genes encode glycosyltransferases (GTs) with unknown substrate specificities. Here we report heterologous expression of one of these enzymes, PttGT43A, a putative IRREGULAR XYLEM9 (IRX9) homologue. Expression trials in Pichia pastoris and insect cells revealed very low levels of accumulation of immunoreactive PttGT43A, whereas transient expression in Nicotiana benthamiana leaves by Agrobacterium infiltration (agroinfiltration) using a viral vector produced substantial amounts of protein that mostly precipitated in the crude pellet. Agroinfiltration induced weak endogenous xylosyltransferase activity in microsomal extracts, and transient PttGT43A expression further increased this activity, albeit only to low levels. PttGT43A may be inactive as an individual subunit, requiring complex formation with unknown partners to display enzymatic activity. Our results suggest that transient co-expression in leaves of candidate subunit GTs may provide a viable approach for formation of an active xylan xylosyltransferase enzymatic complex.