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To shed new light on gene involvement in plant cuticular-wax production, 11 eceriferum (cer) mutants of Arabidopsis having dramatic alterations in wax composition of inflorescence stems were used to create 14 double cer mutants each with two homozygous recessive cer loci. A comprehensive analysis of stem waxes on these double mutants revealed unexpected CER gene interactions and new ideas about individual CER gene functions. Five of the 14 double cer mutants produced significantly more total wax than one of their respective cer parents, indicating from a genetic standpoint a partial bypassing (or complementation) of one cer mutation by the other. Eight of the 14 double cer mutants had alkane amounts lower than both respective cer parents, suggesting that most of these CER gene products play a major additive role in alkane synthesis. Other results suggested that some CER genes function in more than one step of the wax pathway, including those associated with sequential steps in acyl-CoA elongation. Surprisingly, complete epistasis was not observed for any of the cer gene combinations tested. Significant overlap or redundancy of genetic operations thus appears to be a central feature of wax metabolism. Future studies of CER gene product function, as well as the utilization of CER genes for crop improvement, must now account for the complex gene interactions described here.

Insertional mutagenesis of Arabidopsis ecotype C24 was used to identify a novel mutant, designated wax2, that had alterations in both cuticle membrane and cuticular waxes. Arabidopsis mutants with altered cuticle membrane have not been reported previously. Compared with the wild type, the cuticle membrane of wax2 stems weighed 20.2% less, and when viewed using electron microscopy, it was 36.4% thicker, less opaque, and structurally disorganized. The total wax amount on wax2 leaves and stems was reduced by >78% and showed proportional deficiencies in the aldehydes, alkanes, secondary alcohols, and ketones, with increased acids, primary alcohols, and esters. Besides altered cuticle membranes, wax2 displayed postgenital fusion between aerial organs (especially in flower buds), reduced fertility under low humidity, increased epidermal permeability, and a reduction in stomatal index on adaxial and abaxial leaf surfaces. Thus, wax2 reveals a potential role for the cuticle as a suppressor of postgenital fusion and epidermal diffusion and as a mediator of both fertility and the development of epidermal architecture (via effects on stomatal index). The cloned WAX2 gene (verified by three independent allelic insertion mutants with identical phenotypes) codes for a predicted 632-amino acid integral membrane protein with a molecular mass of 72.3 kD and a theoretical pI of 8.78. WAX2 has six transmembrane domains, a His-rich diiron binding region at the N-terminal region, and a large soluble C-terminal domain. The N-terminal portion of WAX2 is homologous with members of the sterol desaturase family, whereas the C terminus of WAX2 is most similar to members of the short-chain dehydrogenase/reductase family. WAX2 has 32% identity to CER1, a protein required for wax production but not for cuticle membrane production. Based on these analyses, we predict that WAX2 has a metabolic function associated with both cuticle membrane and wax synthesis. These studies provide new insight into the genetics and biochemistry of plant cuticle production and elucidate new associations between the cuticle and diverse aspects of plant development.