Identification and characterization of Arabidopsis ECERIFERUM8 (CER8), a gene important for cuticular wax biosynthesis

As one of the major components of the plant cuticle, cuticular waxes play essential roles in many ecophysiological functions, such as resistance to drought and pathogens. Arabidopsis thaliana mutants with abnormal wax load and/or composition are named eceriferum (cer), which are typically identified by their visibly glossy stems. More than 30 cer loci have been identified, but the identification and roles of most of these CER genes are unknown.;By using a combination of forward and reverse genetic approaches, I have identified the CER8 gene as Long-chain Acyl-CoA Synthetase 1 (LACS1). The allelism of CER8 and LACS1 was confirmed by genetic complementation crosses transcript analysis, and gene sequencing. Scanning electron microscopy (SEM) of cer8 mutants showed that they have reduced wax crystal density and lack the tube-shaped wax crystals found on wild-type plants. Gas chromatography analysis of cer8 mutant lines showed that most of the main wax components, including C29 alkanes, C29 ketones, C29 secondary alcohols and C30 primary alcohols are significantly decreased. The total wax load of cer8 null alleles are decreased to approximately half that of the wild type levels. Conversely, C30 free fatty acids are increased by about 15-fold. This is consistent with a defect in a very long-chain acyl CoA synthetase activity specific for C30 fatty acids. The promoter regions of CER8/LACS1 and two other epidermal-expressed LACSs, LACS2 and LACS3 were fused with the GUS reporter gene to analyze the gene expression patterns. LACS1 was found to be epidermal specific, while LACS2 and LACS3 are expressed in the internal cell layers but accumulate to higher levels in the epidermal cell layers. Finally, the substrate specificities of LACS1, -2 and -3 were characterized by using a colourimetric assay with saturated fatty acids of even chain lengths ranging from C16 to C30. LACS1 shows significantly higher activity for C30 free fatty acids than LACS2 and LACS3, which correlates the increased level of C30 free fatty acids in the cer8 mutant alleles. These results suggest that LACS1 and an unknown acyl-CoA thioesterase together provide balanced levels of C30 fatty acyl-CoAs/free fatty acids necessary for the process of cuticular wax biosynthesis.