Abstract
Dimorphecolic acid (9-OH-18:2Delta(10)(trans)(,12)(trans)) is the major fatty acid of seeds of Dimorphotheca species. This fatty acid contains structural features that are not typically found in plant fatty acids, including a C-9 hydroxyl group, Delta(10),Delta(12)-conjugated double bonds, and trans-Delta(12) unsaturation. Expressed sequence tag analysis was conducted to determine the biosynthetic origin of dimorphecolic acid. cDNAs for two divergent forms of Delta(12)-oleic acid desaturase, designated DsFAD2-1 and Ds-FAD2-2, were identified among expressed sequence tags generated from developing Dimorphotheca sinuata seeds. Expression of DsFAD2-1 in Saccharomyces cerevisiae and soybean somatic embryos resulted in the accumulation of the trans-Delta(12) isomer of linoleic acid (18: 2Delta(9)(cis)(,12)(trans)) rather than the more typical cis-Delta(12) isomer. When co-expressed with DsFAD2-1 in soybean embryos or yeast, DsFAD2-2 converted 18:2Delta(9)(cis)(,12)(trans) into dimorphecolic acid. When DsFAD2-2 was expressed alone in soybean embryos or together with a typical cis-Delta(12)-oleic acid desaturase in yeast, trace amounts of the cis-Delta(12) isomer of dimorphecolic acid (9-OH-18:2Delta(10)(trans,)(12)(cis)) were formed from DsFAD2-2 activity with cis-Delta(12)-linoleic acid [corrected]. These results indicate that DsFAD2-2 catalyzes the conversion of the Delta(9) double bond of linoleic acid into a C-9 hydroxyl group and Delta(10)(trans) double bond and displays a substrate preference for the trans-Delta(12), rather than the cis-Delta(12), isomer of linoleic acid. Overall these data are consistent with a biosynthetic pathway of dimorphecolic acid involving the concerted activities of DsFAD2-1 and DsFAD2-2. The evolution of two divergent Delta(12)-oleic acid desaturases for the biosynthesis of an unusual fatty acid is unprecedented in plants.
Highlights
Dimorphecolic acid (9-OH-18:2⌬10trans,12trans) is the major fatty acid of seeds of Dimorphotheca species
Expressed sequence tag analysis was conducted to determine the biosynthetic origin of dimorphecolic acid. cDNAs for two divergent forms of ⌬12-oleic acid desaturase, designated DsFAD2-1 and DsFAD2-2, were identified among expressed sequence tags generated from developing Dimorphotheca sinuata seeds
Identification of Two Divergent FAD2 Polypeptides in D. sinuata Seeds—The enzymes associated with the synthesis of dimorphecolic acid have not been previously identified, and little direct evidence has been presented for its biosynthetic pathway
Summary
X:Y indicates that the fatty acid contains X number of carbon atoms and Y number of double bonds. ⌬z indicates that a double bond is located at the zth carbon atom relative to the carboxyl end of the fatty acid. Given the close taxonomic relation of the Calendula and Dimorphotheca genera, it can be speculated that the C-9 hydroxyl group and trans-⌬10 double bond of dimorphecolic acid arise from modification of the ⌬9 double bond of 18:2⌬9cis,12trans by the activity of an enzyme that is structurally and functionally related to the Calendula ⌬9-fatty acid conjugase. Based on this line of reasoning, the biosynthetic pathway of dimorphecolic acid would involve two specialized enzymes: (i) an enzyme that initially generates the trans-⌬12 double bond of 18:2⌬9cis,12trans and (ii) an enzyme that subsequently converts the ⌬9 double bond of. We demonstrate that DsFAD2-1 and DsFAD2-2 catalyze novel activities that function together to produce dimorphecolic acid in a manner consistent with the biosynthetic pathway proposed above
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