Cloning and That Affects Characterization of CER2, an Arabidopsis Gene Cuticular Wax Accumulation

Cuticular waxes are complex mixtures of very long chain fatty acids and their derivatives that cover plant surfaces. Mutants of the ECERFERUMP (cer2) gene of Arabidopsis condition bright green stems and siliques, indicative of the relatively low abundance of the cuticular wax crystals that comprise the wax bloom on wild-type plants. We cloned the CERP gene via chromosome walking. Three lines of evidence establish that the cloned sequence represents the CERP gene: (1) this sequence is capable of complementing the cer2 mutant phenotype in transgenic plants; (2) the corresponding DNA sequence isolated from plants homozygous for the cer2-2 mutant allele contains a sequence polymorphism that generates a premature stop codon; and (3) the deduced CER2 protein sequence exhibits sequence similarity to that of a maize gene (glossy2) that also is involved in cuticular wax accumulation. The CERP gene encodes a nove1 protein with a predicted mass of 47 kD. We studied the expression pattern of the CERP gene by in situ hybridization and analysis of transgenic Arabidopsis plants carrying a CERP-p-glucuronidase gene fusion that includes 1 .O kb immediately upstream of CERP and 0.2 kb of CERP coding sequences. These studies demonstrate that the CERP gene is expressed in an organ- and tissue-specific manner; CERP is expressed at high levels only in the epidermis of young siliques and stems. This finding is consistent with the visible phenotype associated with mutants of the CERP gene. Hence, the 1.2-kb fragment of the CERP gene used to construct the CERP-P-glucuronidase gene fusion includes all of the genetic information required for the epidermis-specific accumulation of CERP mRNA.