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UBC Theses and Dissertations

Identification and characterization of a poplar ECERIFERUM2-LIKE, a clade II BAHD acyltransferase from hybrid poplar (Populus alba × grandidentata) von Loessl, Michelle


BAHD acyltransferases belong to a large family of enzymes that are involved in the biosynthesis of a wide variety of biologically diverse and important plant compounds. Poplar makes an assortment of different metabolites and therefore has a large number of putative BAHD-acyltransferases, of which only a few have been characterized to date. In this study, an uncharacterized BAHD acyltransferase, with a putative function in either non-canonical monolignol biosynthesis as the poplar p-hydroxybenzoate-CoA monolignol transferase (pHBMT), or in cuticular wax biosynthesis through very long chain fatty acid (VLCFA) elongation as a poplar ECERIFERUM2-LIKE (CER2-LIKE), was studied. To test the function of Populus alba × grandidentata acyltransferase-like (Pa×gACT-like), transgenic Arabidopsis thaliana and hybrid poplar plants mis-regulating the gene, were generated and subsequently analyzed for changes in both lignin p-hydroxybenzoylation and in the cuticular wax composition. No changes in lignin p-hydroxybenzoylation was observed, negating the possibility of Pa×gACT-like being a p-hydroxybenzoate-CoA monolignol transferase. The introduction of Pa×gACT-like into Arabidopsis caused the accumulation of aliphatic wax components 28 carbons in length (C₂₈), and a reduction in C₃₀ wax components, likely due to competition with the native AtCER2 or slight differences in substrate specificity between Pa×gACT-like and AtCER2. In addition, RNAi-suppression of Pa×gACT-like in poplar resulted in a subtle phenotype showing a trend for accumulation of C₂₈ wax components, suggesting an upstream “block” in VLCFA elongation. The shifts in cuticular wax chain length distribution detected in transgenics support a CER2-LIKE role for Pa×gACT-like. Consistent with this, heterologous gene expression in yeast (Saccharomyces cerevisiae) clearly demonstrated that Pa×gACT-like can participate in the elongation of very long chain fatty acids. Co-expression and tissue-specific expression also support a role in cuticular wax biosynthesis, as 30% of genes co-expressed with Pa×gACT-like have predicted functions in lipid biosynthesis/metabolism. Finally, GUS histochemical staining of Pa×gACT-like Prom::GUS poplar transgenics revealed expression in the epidermis of leaf, petiole, and young stem tissue. This study reveals a function for a previously uncharacterized poplar BAHD acyl transferase as a CER2-LIKE protein that functions in the elongation of VLCFAs for cuticular wax production.

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