Dimorphecolic acid (9-OH-18:2Δ10trans,12trans) 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, Δ10,Δ12-conjugated double bonds, and trans Δ12 unsaturation. Expressed sequence tag analysis was conducted to determine the biosynthetic origin of dimorphecolic acid. cDNAs for two divergent forms of Δ12-oleic acid desaturase, designated sFAD2-1 and DsFAD2-2, 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-Δ12 isomer of linoleic acid (18:2Δ9cis,12trans) rather than the more typical cis-Δ12 isomer. When co-expressed with DsFAD2-1 in soybean embryos or yeast, DsFAD2-2 converted 18:2Δ9cis,12trans into dimorphecolic acid. When DsFAD2-2 was expressed alone in soybean embryos or together with a typical cis-Δ12-oleic acid desaturase in yeast, trace amounts of the cis-Δ12 isomer of dimorphecolic acid (9-OH-18:2Δ9cis,12cis) were formed from DsFAD2-2 activity with cis-Δ12-linoleic acid. These results indicate that DsFAD2-2 catalyzes the conversion of the Δ9 double bond of linoleic acid into a C-9 hydroxyl group and Δ10trans double bond and displays a substrate preference for the trans-Δ12, rather than the cis-Δ12, 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 Δ12-oleic acid desaturases for the biosynthesis of an unusual fatty acid is unprecedented in plants. [ABSTRACT FROM AUTHOR]