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

Abstract

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 elongation (FAE) 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 through 7). In the present study, five of these genes were heterologously expressed in yeast and their respective fatty acid profiles determined. PtCER2-likes differentially altered the accumulation of C28 and C30 fatty acids when expressed in the presence of the condensing enzyme AtCER6. Among these, PtCER2-like5 produced the highest levels of C28 fatty acids in yeast and its expression was localized to the epidermis in GUS-reporter poplar lines, consistent with a role in cuticular wax biosynthesis. Complementation of the A. thaliana cer2-5 mutant with PtCER2-like5 increased 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.

Evolution and molecular basis of substrate specificity in a 3-ketoacyl-CoA synthase gene cluster from Populus trichocarpa

“…To test whether PtCER2s could extend the chain length specificity of different poplar KCSs and identify which regions on the KCS are important for this synergistic interaction, we coexpressed a previously characterized PtCER2-like5 with different KCSs and chimeras ( Fig. 6 A ) ( 33 ). When PtCER2-like5 was coexpressed with PtCER6, the product specificity was shifted toward C30 VLCFAs ( Fig.…”

“…The substrate specificity of CER6 has also been shown to be modulated by a binding partner, CER2, which modifies the elongation capability of CER6 to even longer VLCFAs ( 14 , 28 , 29 ). CER2s and their paralogs (termed CER2-likes) are part of the functionally diverse BAHD-acyltransferase family and have been characterized in Arabidopsis, rice, maize, sacred lotus, and poplar ( 28 , 29 , 30 , 31 , 32 , 33 ).…”

Tackling functional redundancy of Arabidopsis fatty acid elongase complexes

“…In addition, several proteins associate with FAE complexes and are required for the completion of the elongation process. These include the CER2-LIKE proteins which are auxiliary enzymatic components of the elongase complexes and obligatory co-factors for specific condensing enzymes, such as KCS6 and KCS5, to enable the production of VLCFA with more than 30 carbons ( Haslam et al., 2015 ; Hegebarth et al., 2017 ; Wang et al., 2017 ; Haslam and Kunst, 2021 ; Gonzales-Vigil et al, 2021 ). Although the mode of action of these proteins remains to be elucidated, it was suggested that they stabilize FAE complexes, enhance their activity or allow the newly elongated acyl-CoA to be presented back to the KCS enzyme for additional elongation cycles.…”

Genome-Wide Analysis of the BAHD Family in Welsh Onion and CER2-LIKEs Involved in Wax Metabolism

“…We also found that the HXXXD motif was present in AfCER2-LIKEs (Figure 5). Previous studies have demonstrated that the HXXXD motif affects the function of CER2 [4,32]. In Welsh onions, the His residue in the HXXXD motif of AfCER2-LIKEs is replaced by Leu, suggesting that the function of AfCER2-LIKE3 may differ from that of AfCER2-LIKE1 and AfCER2- In order to further understand the roles of these genes in adapting to the environment, we analyzed their expression in leaves and stems using RNA-seq.…”