Abstract

Mutations at the fad7 locus of Arabidopsis thaliana (previously called fadD) cause decreased desaturation of dienoic fatty acids in chloroplast lipids in plants grown at elevated temperatures. This suggested that the fad7 locus encodes a chloroplast omega-3 desaturase that catalyzes the desaturation of lipid-linked 18:2 and 16:2 fatty acids. In order to clone the fad7 gene, it was first genetically mapped relative to the flanking restriction fragment length polymorphism markers 4547 and 2488A on chromosome 3, and yeast artificial chromosomes covering the locus were identified. A putative desaturase cDNA clone that was isolated by low stringency heterologous probing with a cDNA for an endoplasmic reticulum-localized omega-3 desaturase (fad3) hybridized to the yeast artificial chromosomes and could not be resolved from the locus by restriction fragment length polymorphism mapping. Expression of the cDNA in transgenic fad7 mutant plants resulted in restoration of wild type fatty acid composition and suppression of a previously observed effect of the fad7 mutation on chloroplast number, indicating genetic complementation. The structural gene contained seven introns within a coding sequence of 1338 base pairs, which encodes a 446-amino acid polypeptide of 51,172 daltons. The amino-terminal region of the fad7 gene product contained a consensus chloroplast transit peptide. Except for the amino-terminal domain, the deduced amino acid sequence of the fad7 gene product had high homology to the fad3 gene product, indicating that fad7 encodes an omega-3 desaturase and that the two genes arose from a common ancestral gene. There was no apparent effect of growth temperature on the steady-state levels of fad7 mRNA in wild type plants.