Understanding the Role of Populus ECERIFERUM2-Likes in the Biosynthesis of Very-Long-Chain Fatty Acids for Cuticular Waxes

Cuticular waxes are derived from very-long-chain fatty acid (VLCFA) precursors made by the concerted action of four enzymes that form the fatty acid (FA) elongation complex. The condensing enzyme of the complex confers specificity to substrates of different chain lengths, yet on its own cannot account for the biosynthesis of VLCFAs longer than 28 carbons (C28). Recent evidence from Arabidopsis thaliana points to a synergistic role of clade II BAHD acyltransferases and condensing enzymes in the elongation of VLCFAs beyond C28. In Populus trichocarpa, clade II is composed of seven uncharacterized paralogous genes (PtCER2-like1–7). In the present study, five of these genes were heterologously expressed in yeast and their respective FA profiles were determined. PtCER2-likes differentially altered the accumulation of C28 and C30 FAs when expressed in the presence of the condensing enzyme AtCER6. Among these, PtCER2-like5 produced the highest levels of C28 FAs in yeast and its expression was localized to the epidermis in β-glucuronidase-reporter poplar lines, consistent with a role in cuticular wax biosynthesis. Complementation of the A. thaliana cer2-5 mutant with PtCER2-like5 increased the levels of C28-derived cuticular waxes at the expense of C30-derived components. Together, these results demonstrate that the role of CER2-likes in cuticular wax biosynthesis is conserved in Populus clade II BAHD acyltransferases.

Arabidopsis ECERIFERUM2-LIKEs Are Mediators of Condensing Enzyme Function

Condensing enzymes catalyze the committed reaction of fatty acid elongation and determine the chain length of fatty acids accepted and produced by the elongation complex. While necessary for the elongation of very-long-chain fatty acids (VLCFAs), identified plant condensing enzymes cannot efficiently produce VLCFAs longer than 28 carbons, which are precursors for the most abundant cuticular waxes of most plant species that have been surveyed. The eceriferum2 (cer2) mutant of Arabidopsis thaliana has a severe wax-deficient phenotype and specifically lacks waxes longer than 28 carbons, but the CER2 protein does not share sequence similarity with condensing enzymes. Instead, CER2 is homologous to BAHD acyltransferases. Heterologous expression in yeast previously demonstrated that CER2, and a small clade of BAHD acyltransferases with high sequence identity to CER2, can extend the chain-length specificity of the condensing enzyme CER6. This biochemical function is distinct from that of the broader BAHD acyltransferase family. The product specificity and physiological functions of individual CER2-LIKE proteins are unique. Here, we demonstrate that CER2 physically interacts with the fatty acid elongase. We cloned chimeric CER2-LIKE proteins and expressed these in yeast cells to identify the features that define the substrate specificities of CER2-LIKEs. We generated homology-based structural models to compare CER2-LIKEs and BAHD acyltransferases. In addition, based on the current phylogenetic analysis of the CER2-LIKE clade, we describe two further Arabidopsis CER2-LIKE genes, CER2-LIKE3 and CER2-LIKE4. We used yeast expression and mutant analysis to characterize these genes. Collectively, these results expand our knowledge of the functions of CER2-LIKEs, the BAHD acyltransferase family and cuticular wax metabolism.