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Roles of CER6 and CER7 proteins in the biosynthesis and regulation of wax production in Arabidopsis thaliana Hooker, Tanya Suzanne


Plant cuticular waxes embed and overlay the cutin matrix of higher plant cuticles. By forming the primary hydrophobic barrier and interface between the plant and the environment, they play important roles in plant responses to abiotic factors, including drought, light and temperature. Cuticular waxes also play important roles in mediating biotic relations, such as plant-insect interactions, as well as fungal and bacterial host and non-host responses. Numerous wax deficient mutants have been isolated from Arabidopsis. The mutations causing wax deficiency identify gene functions required for wax deposition and allow cloning of these genes. These gene functions include biosynthetic enzymes, regulatory factors, and proteins involved in wax secretion. Thus, they are an invaluable.resource for increasing our understanding of wax production. Several wax-related genes have been cloned in recent years. One of these genes is CER6, which encodes an enzyme involved in the first step of wax biosynthesis: the elongation of fatty acids to produce very long chain fatty acid (VLCFA) wax precursors. Since the cer6 mutant has a severe waxless phenotype due to a lesion in a protein early in the wax biosynthetic pathway, we hypothesized that wax deposition could be controlled, at least in part, by the regulation of CER6 expression in plants. To determine the extent of its role in the plant, I investigated its expression pattern in detail. My results indicate that CER6 is transcribed exclusively in epidermal cells throughout shoot development. This expression pattern, and the increase in wax load found in transformed plants over-expressing CER6 confirmed that CER6 is a key enzyme involved in wax synthesis in Arabidopsis shoots. To investigate whether wax deposition in Arabidopsis was influenced by environmental factors, and if this process was to some extent mediated by transcriptional regulation of CER6, I examined CER6 transcript levels and wax loads of plants grown in the absence of light, plants exposed to osmotic stress and different temperature regimes, as well as in wounded plants. I found that light is required for CER6 transcript accumulation, and that osmotic stress, cold temperatures, and ABA induce CER6 transcription. Changes in wax load correlated with CER6 transcript levels in cold-treated plants. However, no correlations could be established for the other conditions tested. To further investigate the regulation of wax deposition in Arabidopsis, I isolated the CER7 gene marked by a mutation in the wax-deficient cer7 mutant, using a positional cloning approach. I first accurately determined the chromosomal location of the cer7 gene using simple sequence length polymorphism (SSLP) marker analysis in an F2 mapping population, and then identified the CER7 gene by complementing the cer7 mutant using genomic fragments from the chromosomal area of interest. I also characterized a Salk Institute T-DNA insertion line with an insertion site 400 bp 5' to the CER7 translational start codon, which represents a second cer7 allele. CER7 is a putative 3'-5' RNAsePH homologue of the yeast Rrp45p, a protein which is one of 10 core proteins forming an RNA processing and degradation complex, the exosome. Transcript analysis of all of the cloned wax-related genes in the cer7 mutant background revealed that none of them was affected by this mutation. Therefore, the CER7 target RNA must represent a factor which has not yet been identified. The results presented in this thesis represent a first concrete step toward elucidating the pathways of regulation of wax production in Arabidopsis, in that I have established the transcriptional regulation pattern of the only known gene coding for an enzyme involved in wax biosynthesis (CER6), and I have identified a new gene (CER7) that is likely to be involved in regulating wax production by influencing the transcript stability of wax-related genes.

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