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The involvement of CER5 in wax secretion of arabidopsis thaliana Pighin, Jamie Ann

Abstract

Plant waxes are found on the surface of plants as a protective layer against many environmental stresses. In Arabidopsis thaliana, they are formed from very long chain fatty acid (VLCFA) precursors that are synthesized in the cytoplasm of epidermal cells. Many eceriferum (cer) mutants of Arabidopsis have been identified that have stems with reduced epicuticular wax. The Cer5 mutant is most interesting because of its unusual cellular phenotype. In the epidermal cells, it contains cytoplasmic extensions into the vacuole with "trilamellar inclusions" within this cytoplasm. Similar sheets are associated with the human neurodegenerative disorder, adrenoleukodystrophy (ALD). In ALD patients, these trilamellar inclusions occur due to a disrupted ABC transporter involved in VLCFA transport through the membrane of peroxisomes. The approximate location of the CER5 gene on chromosome 1 was identified through positional mapping. Two ABC transporter genes were found within this region. Salk T-DNA insertional mutants were obtained with lesions in each of these ABC transporters and one mutant line exhibited a glossy phenotype. A complementation test confirmed that the T-DNA insertion and the cer5 mutation were at the same locus. Molecular complementation of the original Cer5 mutant was done to confirm that the correct gene had been cloned. Gas chromatography was used to quantify the wax load on the surface of Arabidopsis stems in both mutant and transformed lines. To determine the nature of the "trilamellar inclusions", gas chromatography was used to differentiate between the wax content within the epidermal cells, and wax on the exterior of the epidermis. The total wax content inside the epidermal cells of the mutant was similar to wildtype, while the wax on the exterior of the mutant was reduced compared to wild-type. This is consistent with a defect in a transporter, which leads to wax buildup inside the epidermal cells. Staining of mutant epidermal peels using Nile Red, a lipid stain, showed that Cer5 cells fluoresced more brightly than the wild-type cells, confirming the lipidic nature of the inclusions. My results identify the first known function of an ABC transporter in wax export in plants.

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