| Acyl-CoenzymeA synthetases (ACS) catalyze the conversion of free fatty acids to fatty acyl-CoAs. ACS activity was previously localized to the outer envelope of chloroplasts. This envelope ACS activity is responsible for the conversion of all de novo synthesized fatty acids to fatty acyl-CoA molecules which are subsequently exported and act as substrates in many pathways of lipid metabolism.; The Arabidopsis genome contains a family of nine genes shown to exhibit ACS activity against long chain fatty acid substrates (LACS). To identify potential chloroplast LACS isoforms from this family, analysis of RNA expression profiles was done. Two isoforms were identified, LACS9 and LACS2, that showed high levels of expression in young, expanding tissues active in de novo fatty acid synthesis. In vitro chloroplast import assays confirmed that LACS9 and LACS2 were targeted to the chloroplast. For LACS9, further evidence of chloroplast localization was shown by transient expression of a LACS9-GFP fusion, which showed GFP fluorescence in the plastid envelopes.; To study the function of LACS9 and LACS2 in vivo, reverse genetics approaches were used. The lacs9-1 mutant did not have a visible phenotype. However, chloroplasts isolated from lacs9-1 showed only 10% of LACS activity of wild type, which indicated that LACS9 was a major chloroplast isoform. The lacs2-1 mutant was dwarfed and leaves were smaller and fewer in number compared to wild type. Further analysis of the mutant indicated that LACS2 was involved in the synthesis of cuticular compounds. Aspects of the phenotype were common to other mutants with cuticular defects: leaves of the lacs2-1 mutant had alterations in wax load and composition and leaf surfaces supported the germination of wild-type pollen. In addition, cell wall and cutin thickness of leaf cross sections were thinner in lacs2-1. Studies of expression by the LACS2 promoter fused to the GUS gene showed that LACS2 is present exclusively in the epidermis.; This is the first genetic characterization of the role of LACSs, and more specifically chloroplast LACS isoforms, in the plant. The isolation of the knockout mutants has proven to be a useful tool in the analysis of chloroplast LACS function in Arabidopsis. |
