Functional and expression analysis of heteromeric acetyl-CoA carboxylase subunit genes of Arabidopsis
Plant heteromeric acetyl-CoA carboxylase (htACCase) catalyzes the first and committed reaction of de novo fatty acid biosynthesis in plastids. Arabidopsis htACCase consists of five subunits: BCCP-1, BCCP-2, BC, alpha-CT, and beta-CT. They were encoded by CAC1-A, CAC1-B, CAC2, CAC3, and accD genes, respectively. The expression of these five genes was studies by real-time RT-PCR and quantitative western analysis. At the mRNA level, CAC1-A, CAC2, CAC3, and accD genes are expressed at a constant molar ratio of 0.5:1.0:0.2:2.0 across all the organs examined, but the expression pattern of CAC1-B is different. At the protein level, there is no correlation in the accumulation among the five subunits;Analyses using different types of non-denaturing PAGE coupled with western blot analysis with subunit-specific antibodies were performed to study the subunit organization in htACCase complex. These results indicate that the Arabidopsis htACCase is a loose complex that readily dissociates. Analyses with reducing and non-reducing SDS-PAGE revealed the occurrence of homodimers of alpha-CT, and of beta-CT held together by disulfide bond(s). The dimerization was unaffected by illumination;Reverse genetics approaches were used to investigate the individual physiological significance of the two paralogous BCCP-coding genes, CAC1-A and CAC1-B. T-DNA knockout mutant analysis showed that disruption of CAC1-A gene results in embryo lethality, but disruption of CAC1-B gene has no discernible phenotype. In situ hybridization showed that CAC1-A and CAC1-B genes were expressed with similar spatial and temporal patterns during embryo development. This indicates that BCCP-1 and BCCP-2 have non-equivalent physiological roles. CAC1-A antisense plants showed a range of morphological changes, which correlate with the reduction in BCCP-1 accumulation. Similar to CAC1-A, disruption of CAC3 gene also results in the embryo lethal phenotype. The reduction of BCCP-1 results in reduced amount of fatty acids (on per plant basis) in leaves and seeds, but doesn't affect the fatty acid composition. In contrast, loss of BCCP-2 changes neither the amount nor the composition of seed fatty acids. These observations suggest that BCCP-1 is important for htACCase activity in planta, but BCCP-2 is dispensable. Further investigation is needed to elucidate the mechanism of the unidirectional redundancy between BCCP-1 and BCCP-2 subunits.
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