| Similar reverse-genetics approach is applied to investigate the function of two biotin-containing enzymes at the interface between catabolism and anabolism: acetyl-CoA carboxylase (ACCase) and 3-methylcrotonyl-CoA carboxylase (MCCase).;Plastidic ACCase catalyzes the first committed step of de novo fatty acid biosynthesis. Antisense Arabidopsis expressing antisense RNA of CAC1-A, which encodes the biotin carboxyl carrier protein (BCCP) of plastidic ACCase under the direction of CaMV 35S promoter, have been obtained. CAC1-A antisense plants with reduced BCCP possess different degrees of similar morphological changes. These include slow growth, smaller plant size, and crinkly and variegated-yellow vegetative and cauline leaves eventually leading to premature cell death. The severity of the morphological change is correlated with the magnitude of the reduction of BCCP protein. Reduced BCCP expression does not alter mRNA and protein abundance of the other three subunits of ACCase, nor does it change fatty acid composition, but it reduces fatty acid content in each CAC1-A antisense plant. Reduced BCCP expression also causes cellular and subcellular abnormalities and expression changes in genes involved in transcription and translation, anti-oxidation, pathogen and other stress responses, cell division control, and cellular structure.;MCCase is involved in mitochondrial Leu catabolism and several interconnected metabolic pathways: the mevalonate shunt and isoprenoid catabolism. Antisense Arabidopsis expressing antisense RNA of MCC-A, which encodes the biotin-containing subunit of MCCase under the direction of CaMV 35S promoter, have been generated. MCC-A antisense plants express various levels of MCC-A. In some lines, MCC-A mRNA, MCC-A protein, and MCCase activity are all below detection. No morphological changes have been observed in MCC-A antisense plants. Moreover, MCC-A antisense plants accumulate more 14C-propionyl-CoA, an intermediate metabolite of peroxisomal Leu catabolic pathway, than wild type plants, after feeding plants with 14C-Leu. Searching Arabidopsis cDNA library identifies putative branched-chain amino acid aminotransferase in mitochondria, peroxisomes, chloroplasts and cytoplasm, but not putative branched-chain 2-ketoacid dehydrogenase genes in peroxisomes. Expression patterns of genes of potential interest over time in the dark are investigated using Affymetrix microarray. Our study provides evidence supporting the existence of the peroxisomal Leu catabolic pathway, but the establishment of a detailed pathway needs more investigation. |
