| Description: | Acetyl-CoA carboxylase (ACCase) catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA. Malonyl-CoA in plastids is the direct precursor for de novo fatty acid synthesis. In Arabidopsis, plastidic ACCase is a tetrameric enzyme, encoded by three nuclear and one plastidic gene. We postulate that if plastidic ACCase is a key regulatory enzyme, a decrease of plastidic ACCase activity would decrease fatty acid, and thus lipid, biosynthesis, triggering a series of physiological changes. We have generated 42 lines of transgenic Arabidopsis plants with reduced levels of the ACCase biotin carboxyl-carrier (BCC) subunit by introducing an antisense cDNA of the corresponding gene. The transgenic plants have different severities of phenotype reflecting the levels of BCC protein. A typical transgenic plant with a mild phenotype has 75-90% of BCC protein (as compared to wild type plants), grows more slowly, and has a smaller size than the wild-type plants. In these plants, the late vegetative and all cauline leaves are curled and variegated yellow. In the curled leaves, cell size, vacuolar area, and intercellular space are dramatically reduced. The subcellular structure of both chloroplasts and mitochondria is significantly altered. Although seed yield is reduced 10-100 fold, there is no significant change in fatty acid composition or content in seeds harvested from the transgenic plants. We propose to investigate changes in the mRNA profiles in the antisense Arabidopsis plants. Rosette leaves 8-12 of 30-day old Arabidopsis thaliana (ecotype Columbia) will be harvested and polyA RNA will be isolated for microarray analysis for both transgenic and wild type plants. Comparison of the results from the transgenic and wild type plants will enable us to identify the changes in global gene expression at the RNA level. Meanwhile, we are investigating the gene expression at the protein level using two-dimensional electrophoresis (2DE) and MALDI mass spectrometry. Interestingly, our preliminary results show that only the levels of very few proteins have been altered, regardless of the severe phenotype in the transgenic plants. With microarray and 2DE, we would be able to identify gene expression changes at mRNA as well as protein levels in plants under lipid stress. These data will give us a good start to investigate how the morphological changes are generated in the transgenic plants, and shed light on gene regulation networked with plastidic ACCase. |
NASCArrays Information at The BAR
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