Arabidopsis contains nine long-chain acyl-coenzyme a synthetase genes that participate in fatty acid and glycerolipid metabolism

Abstract

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.

Figure 1

Only LACSs contain the eukaryote-type linker domain. A, Schematic comparison of domain structures of various Arabidopsis AMPBP genes. The domain structure of a representative LACS gene (LACS1) is compared with the 4-coumarate-CoA ligase gene At4CL1 and the acetyl-CoA synthetase gene At5g36880. The comparisons were carried out using the GAP program with default parameters (Wisconsin Package Version 10.0, Genetics Computer Group, Madison, WI). The domain nomenclature is described in “Results.” The domain-specific degree of amino acid similarity between LACS1 and the other genes are shown as percentages between each pair of genes. B, Comparison of central regions of a candidate LACS to acetyl-CoA synthetase and 4-coumarate-CoA ligases of Arabidopsis. The amino acid sequences spanning from the C-terminal border of the LS1 domain to the N-terminal border of the LS2 domain were aligned and shaded using the ClustalX and GeneDoc programs. Arrows denote the approximate borders of the linker domain.

Figure 2

Phylogenetic comparison of 11 candidate LACS genes. The deduced full-length amino acid sequences of the genes that contained both the AMPBP signature motif and the linker domain were aligned and displayed as an unrooted nearest neighbor phylogenetic tree using the TreeView program.

Figure 3

LACS enzyme activity measurements from cell-free lysates of overexpressing yeast strains. Gal-induced liquid cultures for each LACS construct were harvested, and spheroplasts prepared and lysed by sonication. Cell-free extracts were used as enzyme sources in in vitro LACS enzyme assays, using 1-[¹⁴C]oleic acid as a substrate. Levels of activity were measured as the numbers of aqueous-soluble counts converted per assay. Each construct was assayed in triplicate. The error bar represents the sd.

Figure 4

Substrate specificity analysis of individual LACS enzymes. All nine Arabidopsis LACS genes were expressed in LACS-deficient E. coli. Membrane fractions were isolated and used as enzyme sources in in vitro enzyme assays using six different radioactive fatty acids. Enzyme levels were normalized as described in “Materials and Methods.” Enzyme activities were measured by liquid scintillation counting and converted into relative units, as described in “Materials and Methods,” to compensate for the differences in the expression levels of each enzyme and specific activities of each fatty acid. Each assay was performed in triplicate. The error bars represent the sds.

Figure 5

Comparison of tissue-specific expression levels of LACS genes. Aliquots of total RNA from each organ or tissue were analyzed for expression of each LACS gene, as well as the actin ACT8 gene as a positive control, by semiquantitative RT-PCR using gene-specific primer pairs. The yield of each product was measured while still in the linear range. R, Root; St, stem; L; leaf; F, flower; DS, developing seed; GS, germinating seedling; S, size marker.