ECERIFERUM2-LIKE Proteins Have Unique Biochemical and Physiological Functions in Very-Long-Chain Fatty Acid Elongation  

Abstract

The extension of very-long-chain fatty acids (VLCFAs) for the synthesis of specialized apoplastic lipids requires unique biochemical machinery. Condensing enzymes catalyze the first reaction in fatty acid elongation and determine the chain length of fatty acids accepted and produced by the fatty acid elongation complex. Although necessary for the elongation of all VLCFAs, known condensing enzymes cannot efficiently synthesize VLCFAs longer than 28 carbons, despite the prevalence of C28 to C34 acyl lipids in cuticular wax and the pollen coat. The eceriferum2 (cer2) mutant of Arabidopsis (Arabidopsis thaliana) was previously shown to have a specific deficiency in cuticular waxes longer than 28 carbons, and heterologous expression of CER2 in yeast (Saccharomyces cerevisiae) demonstrated that it can modify the acyl chain length produced by a condensing enzyme from 28 to 30 carbon atoms. Here, we report the physiological functions and biochemical specificities of the CER2 homologs CER2-LIKE1 and CER2-LIKE2 by mutant analysis and heterologous expression in yeast. We demonstrate that all three CER2-LIKEs function with the same small subset of condensing enzymes, and that they have different effects on the substrate specificity of the same condensing enzyme. Finally, we show that the changes in acyl chain length caused by each CER2-LIKE protein are of substantial importance for cuticle formation and pollen coat function.

Genetic and Biochemical Mechanisms of Pollen Wall Development

“…Genes related to long-chain fatty acids (LCFA) biosynthesis or transport are essential for tryphine formation. Mutations in ECERIFERUM (CER) genes including CER1 [27], CER3/FLP1 [28], and three CER2-LIKE genes [29], as well as in Long-Chain Acyl-CoA Synthetases (LACS) 1 and 4 [26], cause abnormal tryphine and exine. Mutants of phosphoserine phosphatase, which catalyzes the last step of the phosphorylated pathway of serine biosynthesis (PPSB), show normal exine without tryphine [30].…”

Arabidopsis ketoacyl‐CoA synthase 16 (KCS16) forms C36/C38 acyl precursors for leaf trichome and pavement surface wax

“…; Haslam et al . ). Secondly, the CER26‐LIKE protein is known to enhance elongation to C 30 , similar as CER2, and to be mainly expressed in flowers but not leaves (Pascal et al .…”

“…; Haslam et al . ), it seems likely that the KCS16‐containing FAE complex too is associated with a CER2‐LIKE enzyme. Three CER2‐LIKE homologues have been identified and characterized in Arabidopsis (CER2, CER26 and CER26‐LIKE); however, their mechanism or function in enhancing the elongation capacity of FAE complexes remains unknown (Haslam et al .…”

Mid‐infrared spectroscopy is a fast screening method for selecting Arabidopsis genotypes with altered leaf cuticular wax

“…The disruption of the CER6 gene, which encodes a substrate‐specific, rate‐limiting condensing enzyme required for the elongation of VLC fatty acids to C28 in length (Hooker et al, ; Millar et al, ), results in severe wax deficiency as evidenced by both GC and lipid‐related FTIR measurements. CER2 and CER2‐LIKE1 gene products work alongside CER6 to generate C30 and longer VLCFA wax precursors required for the formation of the bulk of cuticular wax in most plant species (Haslam et al, , ). Our GC and FTIR analyses of the cer2‐5 cer2‐like1‐1 mutant confirmed reduced levels of C32 wax components and relatively smaller areas of CH 2 and CH 3 peaks, respectively, and accumulation of C28 and C26 wax products.…”