Cloning of a Temperature-Regulated Gene Encoding a Chloroplast [omega]-3 Desaturase from Arabidopsis thaliana

Abstract

Previous genetic evidence suggested that the fad8 and fad7 genes of Arabidopsis thaliana encode chloroplast membrane-associated 0-3 desaturases. A putative fad8 cDNA was isolated by heterologous hybridization using a gene encoding an endoplasmic reticulum-localized W-3 desaturase (fad3) as a probe. The cDNA encodes a protein of 435 amino acid residues with a molecular mass of 50,134 D. Constitutive expression of the cDNA in transgenic plants of a fad7 mutant resulted in genetic complementation of the mutation, indicating that the fad7 and fad8 gene products are functionally equivalent. Expression of the fad8 cDNA in transgenic plants often resulted in the co-suppression of both the endogenous fad7 and fad8 genes in spite of the fact that these two genes share only about 75% nucleotide identity. In contrast to all other known plant desaturases, including fad7, the steady-state level of fad8 mRNA is strongly increased in plants grown at low temperature. This suggests that the role of fad8 is to provide increased w-3 desaturase activity in plants that are exposed to low growth temperature. The fads-1 mutation created a premature stop codon 149 amino acids from the amino-terminal end of the fad8 open reading frame, suggesting that this mutation results in a complete loss of fad8 activity.The chloroplast membranes of higher plants have unusually high concentrations of tienoic fatty acids, with linolenic (Clk3) or a combination of linolenic and hexadecatienoic (C16:3), making up more than 80% of the fatty acids found in this organelle (Harwood, 1982). These fatty acids are synthesized by sequential insertion of double bonds into derivatives of stearic (Clk0) and hexadecanoic (C,,,,) acids. The formation of the first double bond in 18-carbon fatty acids is generally catalyzed within chloroplasts by one of the few soluble plant desaturases, the stearoyl-acyl carrier protein desaturase (McKeon and Stumpf, 1982). The formation of the second and third double bonds in 18-carbon fatty acids can then ~ ~

Fatty Acid Desaturation during Chilling Acclimation Is One of the Factors Involved in Conferring Low-Temperature Tolerance to Young Tobacco Leaves

“…This observation suggests that an isozyme of the FAD7 desaturase is induced at temperatures between 20 and 26°C and compensates for the deficiency. Recently, a locus (FAD8) encoding such temperature-regulated chloroplast w-3 fatty acid desaturase was identified from A. thaliunu (McConn et al, 1994) and the corresponding cDNA clone was isolated (Gibson et al, 1994). Therefore, our results indicate that an overexpression of the FAD7 gene in tobacco plants leads to the same compositions of fatty acids as found in whole leaves exposed to the temperatures in which endogenous FAD8-type desaturases are induced.…”

“…Another mutation has been identified at a new locus that m,iy correspond to another chloroplast 0-3 fatty acid desaturase (McConn et al, 1994). Moreover, a gene for a temperatureregulated chloroplast 0-3 fatty acid desaturase, w hich has been designated as the FAD8 gene, was isolated from A. thnliuna (Gibson et al, 1994). Thus, at temperaturl2s below 26°C the genes for the FAD8-type desaturases are expressed and DAS are unsaturated actively to produce TAS.…”

The Critical Requirement for Linolenic Acid Is Pollen Development, Not Photosynthesis, in an Arabidopsis Mutant.

“…Leaves of these fad3-2 fad7-1/fad7-2 fadS plants contained only 2% trienoic fatty acids. These supplementary genotypes provide important additional tools in studying the consequences of 18:3 deficiency.The three genetic loci involved in generating the triple mutant are known to be the structural genes encoding three membrane-bound, o>-3 desaturases in Arabidopsis (Arondel et al, 1992;Iba et al, 1993; Yadav et al, 1993;Gibson et al, 1994). Our results indicate that these are the only quantitatively significant desaturases contributing to 16:2 and 18:2 desaturation in leaf tissues.…”

The effects of elevated atmospheric CO2 on lipid metabolism in leaves from mature wheat (Triticum aestivum cv. Hereward) plants

“…For example, probing the wheat tissues for expression of different desaturase genes would certainly be possible as appropiate cDNAs from other plants are already available (e.g. Gibson et al 1994;Falcone et al 1994;see Murata & Wada 1995).…”