RESUMO
Acyl-coenzyme A (CoA) synthetases (ACSs, EC 6.2.1.3) catalyze the formation of fatty acyl-CoAs from free fatty acid, ATP, and CoA. Essentially all de novo fatty acid synthesis occurs in the plastid. Fatty acids destined for membrane glycerolipid and triacylglycerol synthesis in the endoplasmic reticulum must be first activated to acyl-CoAs via an ACS. Within a family of nine ACS genes from Arabidopsis, we identified a chloroplast isoform, LACS9. LACS9 is highly expressed in developing seeds and young rosette leaves. Both in vitro chloroplast import assays and transient expression of a green fluorescent protein fusion indicated that the LACS9 protein is localized in the plastid envelope. A T-DNA knockout mutant (lacs9-1) was identified by reverse genetics and these mutant plants were indistinguishable from wild type in growth and appearance. Analysis of leaf lipids provided no evidence for compromised export of acyl groups from chloroplasts. However, direct assays demonstrated that lacs9-1 plants contained only 10% of the chloroplast long-chain ACS activity found for wild type. The residual long-chain ACS activity in mutant chloroplasts was comparable with calculated rates of fatty acid synthesis. Although another isozyme contributes to the activation of fatty acids during their export from the chloroplast, LACS9 is a major chloroplast ACS.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cloroplastos/metabolismo , Coenzima A Ligases/genética , Ácidos Graxos/metabolismo , Acil Coenzima A/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Cloroplastos/enzimologia , Coenzima A Ligases/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Cebolas/genética , Cebolas/metabolismo , Pisum sativum/citologia , Pisum sativum/metabolismo , Fenótipo , Epiderme Vegetal/genética , Epiderme Vegetal/metabolismoRESUMO
Long-chain acyl-coenzyme A (CoA) synthetases (LACSs) activate free fatty acids to acyl-CoA thioesters and as such play critical roles in fatty acid metabolism. This important class of enzymes factors prominently in several fatty acid-derived metabolic pathways, including phospholipid, triacylglycerol, and jasmonate biosynthesis and fatty acid beta-oxidation. In an effort to better understand the factors that control fatty acid metabolism in oilseeds, we have sought to identify and characterize genes that encode LACSs in Arabidopsis. Nine cDNAs were identified, cloned, and tested for their ability to complement a LACS-deficient strain of yeast (Saccharomyces cerevisiae). Seven of the nine successfully restored growth, whereas two cDNAs encoding putative peroxisomal isoforms did not. Lysates from yeast cells overexpressing each of the nine cDNAs were active in LACS enzyme assays using oleic acid as a substrate. The substrate specificities of the enzymes were determined after overexpression in LACS-deficient Escherichia coli. Most of the LACS enzymes displayed highest levels of activity with the fatty acids that make up the common structural and storage lipids in Arabidopsis tissues. Analysis of the tissue-specific expression profiles for these genes revealed one flower-specific isoform, whereas all others were expressed in various tissues throughout the plant. These nine cDNAs are thought to constitute the entire LACS family in Arabidopsis, and as such, will serve as powerful tools in the study of acyl-CoA metabolism in oilseeds.
