UBC Theses and Dissertations
Localized post-transcriptional gene silencing of the very long chain fatty acid condensing enzyme, CER6, and analysis of transcript accumulation in the anthers of developing flower buds of Arabidopsis thaliana Houlahan, Nora Kathleen
In Arabidopsis thaliana, both the stem wax and the pollen lipids are generated from very long chain fatty acid (VLCFA) precursors. A mutation in the gene encoding CER6, a condensing enzyme involved in VLCFA synthesis, results in plants with alterations in both the stem wax and the pollen coat lipid profiles. Phenotypically these plants lack epicuticular wax crystals on their stems and are conditionally male sterile. When grown under conditions of low humidity, the pollen of cer6 plants does not hydrate on a receptive stigma, although hydration will occur under high humidity growth conditions. This conditional male sterility can be exploited for use in breeding systems. Pollination of cer6 plants as the female parent will ensure no self-pollinated contaminants and the line can be easily propagated for future use through growth in high humidity. Alterations in stem wax profile are, however, not desirable as they may negatively influence the fitness of the plant. To address this problem, an RNAi construct was designed to induce localized silencing of the CER6 gene in the tissues where the pollen coat is produced. Transgenic plants expressing this silencing construct displayed a wildtype stem wax load and exhibited conditional male sterility. Analysis of RNA extracted from flower buds of the TI generation showed a decrease in CER6 expression at stage 12 of floral development in plants that were phenotypically conditionally male sterile. Analysis of the subsequent T2 and T3 generations revealed a reversion to a wildtype fertile phenotype in all of the initially silenced lines. Examination of CER6 transcript levels confirmed that this fertility was correlated to wildtype levels of expression at stage 12 of floral development. This loss of silencing was not the result of an aberrant transgene transcript that was unable to initiate silencing, as no such transcript could be detected in any of the plants examined. Rather, methylation of the transgene promoter was observed, indicating the transcriptional silencing of transgene expression.
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