Abstract
Divergent forms of the plant Delta(12)-oleic-acid desaturase (FAD2) have previously been shown to catalyze the formation of acetylenic bonds, epoxy groups, and conjugated Delta(11),Delta(13)-double bonds by modification of an existing Delta(12)-double bond in C(18) fatty acids. Here, we report a class of FAD2-related enzymes that modifies a Delta(9)-double bond to produce the conjugated trans-Delta(8),trans-Delta(10)-double bonds found in calendic acid (18:3Delta(8trans,10trans,12cis)), the major component of the seed oil of Calendula officinalis. Using an expressed sequence tag approach, cDNAs for two closely related FAD2-like enzymes, designated CoFADX-1 and CoFADX-2, were identified from a C. officinalis developing seed cDNA library. The deduced amino acid sequences of these polypeptides share 40-50% identity with those of other FAD2 and FAD2-related enzymes. Expression of either CoFADX-1 or CoFADX-2 in somatic soybean embryos resulted in the production of calendic acid. In embryos expressing CoFADX-2, calendic acid accumulated to as high as 22% (w/w) of the total fatty acids. In addition, expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was accompanied by calendic acid accumulation when induced cells were supplied exogenous linoleic acid (18:2Delta(9cis,12cis)). These results are thus consistent with a route of calendic acid synthesis involving modification of the Delta(9)-double bond of linoleic acid. Regiospecificity for Delta(9)-double bonds is unprecedented among FAD2-related enzymes and further expands the functional diversity found in this family of enzymes.
Highlights
The common polyunsaturated fatty acids of plant seed oils contain cis-double bonds that are separated by a methylene group
Identification of Divergent FAD2 cDNAs in C. officinalis Seeds—An expressed sequence tag (EST) approach was used to determine the biosynthetic origin of calendic acid in C. officinalis seeds
DNA sequences were obtained from the 5Ј-ends of Ͼ3000 randomly selected cDNAs from a C. officinalis developing seed library. From this pool of ESTs, 12 cDNAs that encode FAD2-related polypeptides were identified by BLAST homology. Five of these cDNAs corresponded to polypeptides that were more closely related to the “typical” plant FAD2 that is associated with the cis-⌬12-desaturation of oleic acid
Summary
We have recently demonstrated that the conjugated trans⌬11- and trans-⌬13-double bonds of ␣-eleostearic and ␣-parinaric acids in seeds of Momordica charantia and Impatiens balsamina, respectively, are synthesized by divergent forms of the ⌬12-oleic-acid desaturase (FAD2; oleate desaturase, EC 1.3.1.35), which we have termed “conjugases” (4) These enzymes catalyze the conversion of an existing cis-⌬12-double bond into conjugated trans-⌬11- and trans-⌬13-double bonds (4, 5). The synthesis of ␣-parinaric acid in I. balsamina seeds arises from the conjugase-catalyzed modification of the cis-⌬12-double bond of ␣-linolenic acid (4) These reactions use fatty acids bound to phosphatidylcholine as substrates (5), as has been shown for other FAD2-type enzymes (6). Expression of either cDNA in somatic soybean embryos results in the accumulation of calendic acid These findings demonstrate that FAD2-type enzymes can catalyze the modification of the ⌬12-position, and the ⌬9-position of fatty acid substrates. We show that calendic acid accumulation in somatic soybean embryos is not accompanied by large increases in oleic acid content, which is in contrast to the phenotype generally observed with the expression of other divergent FAD2 enzymes in transgenic plants (4, 13, 14)
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