Article Figures & Data
Figures
- Fig. 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.
- Fig. 2.
Phylogenetic comparison of 11 candidateLACS 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.
- Fig. 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.
- Fig. 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.
- Fig. 5.
Comparison of tissue-specific expression levels ofLACS 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.
Tables
- Table I.
Summary of Genbank accession nos., chromosomal locations, and EST profiles of cloned LACS genes
Gene Genbank Accession No. Chromosome/MIPS Code Corresponding ESTs LACS1 AF503751 Chromosome 2, At2g47240 Developing seed: eight Green siliques: seven Roots: two Flower buds/inflorescences: two LACS2 AF503752 Chromosome 1, At1g49430 Developing seed: one Green siliques: four Roots: one Flower buds/inflorescences: one LACS3 AF503753 Chromosome 1, At1g64400 Rosette leaves: one Liquid-cultured seedlings: one LACS4 AF503754 Chromosome 4, At4g23850 Green siliques: two LACS5 AF503755 Chromosome 4, At4g11030 None LACS6 AF503756 Chromosome 3, At3g05970 Green siliques: one Roots: one LACS7 AF503757 Chromosome 5, At5g27600 Developing seed: two Roots: one LACS8 AF503758 Chromosome 2, At2g04350 Roots: one Etiolated hypocotyls: one LACS9 AF503759 Chromosome 1, At1g77590 Developing seed: two Green siliques: two Roots: three
