Dimorphecolic Acid Is Synthesized by the Coordinate Activities of Two Divergent Δ12-Oleic Acid Desaturases*

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 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-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.

Dimorphecolic acid (9-OH-18:2⌬ 10trans,12trans ) 1 is an unusual C 18 fatty acid that can comprise more than 60% of the seed oil of Dimorphotheca species (1). This fatty acid has received attention because of its potential value for industrial applica-tions. For example, the chemical functionalities resulting from its C-9 hydroxyl group and conjugated ⌬ 10 ,⌬ 12 double bonds make dimorphecolic acid useful in the manufacture of paints, inks, lubricants, plastics, and nylon (2,3).
The biosynthetic pathway of dimorphecolic acid has not been previously determined. This fatty acid would appear to be of complex biosynthetic origin given the presence of three structural features that are not typically found in plant fatty acids: (i) a C-9 hydroxyl group, (ii) conjugated ⌬ 10 ,⌬ 12 double bonds, and (iii) a trans-⌬ 12 double bond. With regard to the trans-⌬ 12 double bond, Morris and Marshall (4) reported nearly 40 years ago that the unusual ⌬ 9cis,12trans isomer of linoleic acid (18:2) comprises more than 1% of the fatty acids of Dimorphotheca sinuata seeds. Based on this finding, these researchers proposed that 18:2⌬ 9cis,12trans is the precursor of dimorphecolic acid rather than the more commonly occurring 18:2⌬ 9cis,12cis . This initial step in the synthesis of dimorphecolic acid would therefore require an unusual enzymatic activity that generates a trans-⌬ 12 double bond instead of the cis-⌬ 12 double bond that is normally formed by the ⌬ 12 -oleic acid desaturase (FAD2) 2 in plants.
When considering the possible biosynthetic origin of the C-9 hydroxyl group and the ⌬ 10 double bond of dimorphecolic acid, it should be noted that the Dimorphotheca genus is taxonomically closely related to the Calendula genus in the plant kingdom. Both genera are members of the Calendulae tribe of the Asteraceae family. Seeds of Calendula sp. produce large amounts of calendic acid (18:3⌬ 8trans,10trans,12cis ), an unusual conjugated fatty acid that has some structural similarity to dimorphecolic acid (5). It has been previously shown that calendic acid is formed by the conversion of ⌬ 9 double bond of linoleic acid to conjugated ⌬ 8 ,⌬ 10 double bonds by the activity of a divergent form of FAD2 (6,7). This enzyme, which has been termed a "⌬ 9 -fatty acid conjugase," is believed to catalyze the removal of hydrogen atoms from the C-8 and C-11 atoms that flank the ⌬ 9 double bond of linoleic acid (8). 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 * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  18:2⌬ 9cis,12trans into a C-9 hydroxyl group and ⌬ 10trans double bond to form dimorphecolic acid.
In this study, an expressed sequence tag (EST) analysis of developing D. sinuata seeds was conducted to provide direct evidence for the biosynthetic origin of dimorphecolic acid. This functional genomic approach has proven to be a powerful method for the identification of enzymes that are involved in the synthesis of unusual fatty acids in plants, including ⌬ 9 -and ⌬ 12 -fatty acid conjugases (6,9), cyclopropane fatty-acid synthase (10), and a cytochrome P450 ⌬ 12 -expoxygenase (11). As described here, EST analysis revealed the occurrence of two structurally divergent forms of FAD2 in D. sinuata seeds that were designated DsFAD2-1 and DsFAD2-2. 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.

EXPERIMENTAL PROCEDURES
Expressed Sequence Tag Analysis of Developing D. sinuata Seeds-Total RNA was isolated from developing seeds of D. sinuata DC. (African daisy) according to the method described by Jones et al. (12). Poly(A) ϩ RNA enrichment and cDNA library construction were performed as described previously (6,9). The cDNA inserts were cloned directionally in the EcoRI and XhoI sites of pBluescript II SK(ϩ). The resulting plasmid library was propagated in Escherichia coli DH10B cells (Invitrogen). Plasmid DNA was prepared from 2,669 randomly selected colonies from the D. sinuata cDNA, and partial nucleotide sequence was obtained from the 5Ј ends of the resulting plasmid as described previously (6,9). Tentative identification of polypeptides corresponding to the sequenced cDNAs was determined by bioinformatic analysis of translated 5Ј sequences using the National Center for Biotechnology Information (NCBI) BLASTX program. Full-length cDNAs for two structurally divergent classes of FAD2, designated DsFAD2-1 and DsFAD2-2, were identified from the EST analysis and were characterized further as described below. The GenBank TM accession numbers for the DsFAD2-1 and DsFAD2-2 cDNAs are AY494986 and AY494985, respectively.
Expression of DsFAD2-1 in Saccharomyces cerevisiae-A full-length cDNA for DsFAD2-1 from the EST analysis above was linked as an EcoRI/XhoI fragment to the GAL1 promoter of the yeast expression vector pYES2 (Invitrogen). The resulting plasmid and the pYES2 vector lacking cDNA insert were introduced into S. cerevisiae YPH499 cells by lithium acetate-mediated transformation (13). Transformed cells were selected for their ability to grow on medium lacking uracil. Single colonies from the transformed cells were grown for 2 days at 28°C in medium consisting of 0.08% (w/v) complete supplement mixture without uracil (CSM-URA, BIO101), 0.17% (w/v) yeast nitrogen base without amino acids (Difco), 0.5% (w/v) ammonium sulfate, 5% glycerol, and 0.5% dextrose. Cells were then washed twice in the same medium except that the glycerol and dextrose were replaced with galactose, which was added at a final concentration of 2% (w/v). The cells were diluted to A 600 Ϸ 0.2 in the galactose-containing medium and grown with shaking (310 rpm) for 24 h at 28°C and then shifted to 22°C until the cells reached A 600 Ϸ 4. The cells were then collected by centrifugation, and fatty acid methyl esters were prepared from the cell pellets as described below.
As a control for these experiments, a cDNA for a cis-⌬ 12 -oleic acid desaturase from Euphorbia lagascae (GenBank TM accession number AY486148) was expressed in S. cerevisiae under the growth conditions described above. The E. lagascae cDNA was linked to the GAL10 promoter of the expression vector pESC-URA (Stratagene) in the NotI/SacI restriction sites.
DsFAD2-1 and DsFAD2-2 were co-expressed in S. cerevisiae by use of the pESC-HIS vector (Stratagene), which contains separate GAL1 and GAL10 promoters for expression of two genes. A full-length cDNA for DsFAD2-2 from the EST analysis was linked as a BamHI/XhoI fragment to the GAL1 promoter of pESC-HIS. The open reading frame of DsFAD2-1 was subsequently linked as a NotI fragment to the GAL10 promoter of the pESC-HIS plasmid containing the DsFAD2-2 cDNA. For this cloning step, the open reading frame of the DsFAD2-1 cDNA was amplified by PCR from a full-length cDNA identified in the EST study using Pfu polymerase (Stratagene). The oligonucleotides used for PCR were: 5Ј-TATGCGGCCGCAAATGGGAGCAGGAGGTTG-3Ј (sense) and 5Ј-TTTGCGGCCGCATTACATCTTATTCTTGTACC-3Ј (antisense). (Note that the underlined sequences correspond to the added NotI restriction sites.) The product was subcloned into the vector pCR-Script AMP SK(ϩ) (Stratagene) prior to introduction in the yeast expression vector. A pESC-HIS-based plasmid was also constructed that contained the cDNA for the E. lagascae cis-⌬ 12 -oleic acid desaturase linked as a NotI-SacI fragment to the GAL10 promoter and the Ds-FAD2-2 cDNA linked as a BamHI/XhoI fragment to the GAL1 promoter. pESC-HIS-derived plasmids containing the DsFAD2-2 cDNA alone or in combination with the DsFAD2-1 cDNA or E. lagascae FAD2 cDNA were transformed into S. cerevisiae and grown as described.
Expression of DsFAD2-1 and DsFAD2-2 in Soybean Somatic Embryos-For expression in somatic embryos of soybean (Glycine max (L.) Merrill cv. Jack), the cDNAs for DsFAD2-1 and DsFAD2-2 were linked to promoter for the ␣Ј-subunit of ␤-conglycinin gene in the vector pKS67 (9). This promoter confers strong seed-specific expression of transgenes. The DsFAD2-1 and DsFAD2-2 cDNAs were introduced into pKS67 as NotI fragments following PCR amplification. The open reading frames of DsFAD2-1 and DsFAD2-2 were amplified by PCR from corresponding full-length cDNAs identified in the EST analysis described above. The open reading frame of DsFAd2-1 was amplified by use of the primers described above. The DsFAD2-2 open reading frame was amplified by using the oligonucleotides 5Ј-TGCGGCCGCAATGGGTGGAGGGATG-GGAGCATCTGAG-3Ј (sense) and 5Ј-TAGCGGCCGCTGATTAATCAA-GTCTTAG-3Ј (antisense). The PCR products were subcloned into the intermediate vector pCR-Script AMP SK(ϩ) (Stratagene) according to the manufacturer's protocol. The DsFAD2-1-and DsFAD2-2-derived PCR products were then ligated as NotI fragments into the corresponding site of pKS67. In experiments involving the co-expression of Ds-FAD2-1 and DsFAD2-2 in soybean somatic embryos, the DsFAD2-1derived NotI fragment was linked to the promoter of the gene for the ␣Ј-subunit of ␤-conglycinin in the previously described vector pKS17 (14). This soybean expression vector is identical to pKS67 except that it lacks a hygromycin resistance marker for selection of transgenic events.
Gene fusions of the DsFAD2-1 or DsFAD2-2 cDNAs with the ␤-conglycinin promoter and phaseolin 3Ј non-translated region in vector pKS67 were introduced into soybean somatic embryos by using particle bombardment (15). Experiments were also conducted in which the DsFAD2-2 cDNA in pKS67 was co-transformed with the DsFAD2-1 cDNA in pKS17. In these experiments, the DsFAD2-1-and DsFAD2-2containing plasmids were co-transformed at a molar ratio of 10:1 (Ds-FAD2-1:DsFAD2-2) of the two expression constructs. A similar cotransformation methodology has been reported previously (14). Transgenic embryos were selected by hygromycin resistance conferred by the marker gene for hygromycin phosphotransferase in pKS67. Hygromycin-resistant embryos were propagated to maturity, and expression of the DsFAD2-1 and DsFAD2-2 transgenes was confirmed by using previously described protocols (6,9).
Fatty Acid Analysis of S. cerevisiae and Soybean Somatic Embryos-Fatty acid methyl esters were prepared from S. cerevisiae cultures by direct transesterification of cell pellets in sodium methoxide/methanol (6). The fatty acid methyl esters were then analyzed by gas chromatography (GC) using an Agilent 6890 chromatograph fitted with a DB-23 column (30-m ϫ 0.25-mm inner diameter, 0.25-m film; Agilent). The oven temperature was programmed from 185°C (2-min hold) to 225°C at 5°C/min, and eluted fatty acid methyl esters were detected by flame ionization. The retention time of the 18:2 methyl ester produced by DsFAD2-1-expressing cells was compared with that of a standard mixture of cis-trans isomers of 18:2⌬ 9,12 methyl ester (Sigma). In addition, structural analysis of fatty acid methyl esters from yeast extracts was conducted by use of GC-mass spectrometry (MS) as described below for the analysis of soybean fatty acid methyl esters.
The double bond positions of 16:2 and 18:2 isomers produced in S. cerevisiae were determined by GC-MS following conversion of fatty acids to diethylamide derivatives. Free fatty acids were initially prepared by saponification of cell pellets from S. cerevisiae cultures (grown as described above) in 1 ml of 0.6 N potassium hydroxide in methanol. Following a 1-h incubation at 70°C, free fatty acids were extracted by partitioning the reaction with the addition of 0.9 ml of 1 N hydrochloric acid and 1 ml of chloroform. Fatty acids were recovered in the chloroform phase, dried under nitrogen, and then converted into diethylamide derivatives using the method described by Nilsson and Liljenberg (16). The resulting fatty acid diethylamide derivatives were analyzed by GC-MS using an HP6890 interfaced with a HP5973 (Agilent) mass selective detector. Sample components were resolved with a DB-23 column, and the oven temperature was programmed from 185°C (2-min hold) to 235°C at 5°C/min.
Fatty acid methyl esters were prepared from soybean somatic embryos by transesterification in 1% (w/v) sodium methoxide in methanol as described previously (6,9). The recovered fatty acid methyl esters were analyzed by GC with an Omegawax 320 column (30-m ϫ 0.32-mm inner diameter; Supelco). The oven temperature was programmed from 185°C (4-min hold) to 215°C at a rate of 5°C/min and then to 240°C at 20°C/min (1-min hold). Fatty acid methyl esters were also analyzed by GC-MS using the instrument described above fitted with an HP-INNO-Wax column. The oven temperature was programmed from 180°C (3.5-min hold) to 215°C at a rate of 2°C/min (2-min hold) and then to 230°C at 10°C/min.
For analyses of yeast cells and soybean embryos transformed with DsFAD2-2, the recovered fatty acid methyl esters were dried under nitrogen and reacted with 50 -100 l of the silylating reagent bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane (99:1, v/v) (Supelco) to convert the hydroxyl group of dimorphecolic acid to a trimethylsilyl (TMS) ether derivative for GC and GC-MS analyses (11). Samples were incubated at 70°C for 30 min. The samples were then dried under nitrogen and resuspended in heptane for GC and GC-MS analyses as described above.
Of note, our identification of cis-trans orientations of ⌬ 12 double bonds of 16:2 and 18:2 methyl esters (as described under "Results") was consistent with the known chromatographic properties of the different GC columns used in the analyses of S. cerevisiae and soybean extracts. For example, the trans isomer of a given fatty acid methyl ester elutes prior to the cis isomer on the 50% cyanopropyl, methylpolysiloxane phase of a DB-23 column (17) as was observed in the analysis of S. cerevisiae extracts (see Fig. 2). Conversely a cis isomer elutes before the corresponding trans isomer on the polyethylene glycol phase of OmegaWax 320 and HP-INNOWax columns (17) as was observed in the analysis of soybean extracts (see Fig. 3).

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. To provide clues for the biosynthetic origin of dimorphecolic acid, an EST analysis of developing D. sinuata seeds was conducted. From the sequences of 2,669 randomly selected cDNAs, 12 ESTs for FAD2-like polypeptides were identified. This was of particular interest because divergent members of the FAD2 family catalyze the synthesis of a number of unusual fatty acids, including those that, like dimorphecolic acid, contain hydroxyl groups and conjugated double bonds (18). The 12 D. sinuata ESTs included five ESTs that appeared to encode a functionally divergent form of FAD2, designated DsFAD2-1, and three ESTs that appeared to encode a second divergent form of FAD2, designated DsFAD2-2. The remaining four ESTs appeared to encode typical cis-⌬ 12 -oleic acid desaturases. The putative identification of D. sinuata FAD2 ESTs as functionally "typical" or "divergent" was based on properties of their deduced amino acid sequences. The primary structures of FAD2s contain three His-rich domains or "boxes" that are believed to coordinate active site diiron atoms (18,19). The consensus sequence of the first His box is HECGHH. In all members of the FAD2 family that function as cis-⌬ 12 -oleic acid desaturases, this box is preceded by Ala. However, all known enzymes of this family that contain Gly in this position catalyze alternative reactions such as fatty acid hydroxylation and epoxygenation (20 -22). The amino acid sequences of both DsFAD2-1 and DsFAD2-2 contain a Gly residue preceding the first His box. Based on this, we hypothesized that DsFAD2-1 and DsFAD2-2 do not function as typical cis-⌬ 12 -oleic acid desaturases. The other four D. sinuata ESTs identified as cis-⌬ 12 -oleic acid desaturases had Ala preceding the first His box and other sequence elements consistent with the cis-⌬ 12 -oleic acid desaturase functional class of FAD2 enzymes (23).
Interestingly the amino acid sequences of DsFAD2-1 and DsFAD2-2 share only 48% identity. DsFAD2-1 is most closely related to a cis-⌬ 12 -oleic acid desaturase from Helianthus annuus and shares ϳ60 -75% amino acid sequence identity with all known cis-⌬ 12 -oleic acid desaturases. This polypeptide also shares 63-67% identity with FAD2-type fatty acid hydroxylases and ⌬ 12 -fatty acid conjugases from Aleurites fordii (23) and Punica granatum (24) but shares Յ55% identity with FAD2 epoxygenases, acetylenases, and ⌬ 9 -fatty acid conjugases. In addition, the amino acid sequence of DsFAD2-1 does not display a distinct phylogenetic relationship with those of any specific FAD2 functional class (Fig. 1). As a result, it was not possible to predict the enzymatic function of DsFAD2-1 from its primary structure alone. DsFAD2-2, by contrast, shares Յ55% identity with all members of the FAD2 family except the ⌬ 9 -fatty acid conjugases from Calendula officinalis (6, 7). DsFAD2-2 and the C. officinalis ⌬ 9 -fatty acid conjugases share ϳ75% amino acid sequence identity, and these polypeptides display a close phylogenetic relationship (Fig. 1). The  Functional Characterization of DsFAD2-1-Enzymes of the FAD2 family have typically proven recalcitrant to direct assay (19). This is likely due, in part, to their association with membranes. The function of FAD2 enzymes instead has been determined by monitoring for novel fatty acid products that accumulate upon their expression in a heterologous host. S. cerevisiae has proven to be a useful system for the functional characterization of newly identified FAD2 enzymes (6,7,(23)(24)(25). In addition, we have previously used soybean somatic embryos for analysis of FAD2 enzymes (9,11). This tissue has proven to be a predictive model system for seed-specific phenotypes, and transgenes can be expressed to high levels in soybean somatic embryos by use of promoters for seed storage protein genes (26).
The function of DsFAD2-1 was initially assessed by expression in S. cerevisiae. Two novel fatty acids, identified by GC-MS as 16:2 and 18:2, were detected upon expression of the Ds-FAD2-1 cDNA (Fig. 2B and Table I). The double bonds of these fatty acids were determined to be at the ⌬ 9 and ⌬ 12 positions based on GC-MS analysis of diethylamide derivatives (data not shown). The GC retention times of the 16:2 and 18:2 methyl esters, however, were different from those of 16:2⌬ 9cis,12cis and 18:2⌬ 9cis,12cis methyl esters from yeast cells expressing a typical cis-⌬ 12 -oleic acid desaturase (Fig. 2C). The 18:2 methyl ester from DsFAD2-1-expressing cells instead displayed a retention time identical to that of methyl 18:2⌬ 9cis,12trans in a standard mixture of methyl 18:2 isomers (Fig. 2D). Based on these chromatographic and mass spectral data, it was concluded that DsFAD2-1 functions as trans-⌬ 12 desaturase, and when expressed in S. cerevisiae, this enzyme is able to convert oleic acid (18:1⌬ 9cis ) into 18:2⌬ 9cis,12trans . It is presumed that the 16:2 formed by DsFAD2-1 is the ⌬ 9cis,12trans isomer that arises from the trans-⌬ 12 desaturation of palmitoleic acid (16: 1⌬ 9cis ). Although ⌬ 9cis,12trans isomers were the predominant form of 16:2 and 18:2 in cells expressing DsFAD2-1 (Table I), ⌬ 9cis,12cis isomers of these fatty acids were detectable at amounts of Ͻ0.2% of the total fatty acids of yeast extracts. This observation suggests that DsFAD2-1 may have a very limited ability to also catalyze cis-⌬ 12 desaturation.
Consistent with the results obtained from yeast, expression of DsFAD2-1 in soybean somatic embryos yielded a novel 18:2 isomer (Fig. 3B). The methyl ester of this isomer displayed a GC retention time that was identical to that of a methyl 18:2 isomer from D. sinuata seed extracts. This isomer has previously been identified as the 18:2⌬ 9cis,12trans (4) (Fig. 3C). The 18:2⌬ 9cis,12trans isomer accounted for 15% of the fatty acids of embryos transformed with the DsFAD2-1 cDNA (Table II). In addition to the production of 18:2⌬ 9cis,12trans , the expression of DsFAD2-1 resulted in a reduction in the content of the 18: 2⌬ 9cis,12cis isomer relative to the nontransformed embryos (Table II). This was likely due to competition of DsFAD2-1 with the native cis-⌬ 12 -oleic acid desaturase of soybean embryos for the available pool of oleic acid, the substrate for both enzymes. Overall the results from yeast and soybean expression indicate that DsFAD2-1 functions primarily as a trans-⌬ 12 -oleic acid desaturase for the conversion of 18:1⌬ 9cis into 18:2⌬ 9cis,12trans .
Functional Characterization of DsFAD2-2-The function of DsFAD2-2 was initially characterized by expression in soybean somatic embryos. Fatty acid methyl esters prepared from the soybean embryos were reacted with a silylating reagent to facilitate the GC analysis of any hydroxy fatty acids, such as dimorphecolic acid, that might be formed by DsFAD2-2 activity. This reagent converts hydroxyl residues into TMS-ether derivatives. The mass spectrum of the TMS derivative of dimorphecolic acid methyl ester has an abundant 225 m/z ion. This diagnostic ion results from fragmentation at the C-8 and C-9 atoms (Fig. 4A). Using GC-MS, the 225 m/z ion can be extracted from total ion chromatograms of soybean embryo fatty acid methyl esters to provide a very sensitive means of detecting dimorphecolic acid production. Through the use of this method, two fatty acid methyl esters with mass spectra identical to that of the TMS derivative of methyl dimorpheco-  late were detected in extracts from soybean embryos expressing DsFAD2-2 (Fig. 5A). Neither fatty acid methyl ester was detected in extracts from non-transformed soybean embryos. The less abundant of the two peaks from DsFAD2-2-expressing embryos had a retention time identical to that of the TMS derivative of methyl dimorphecolate from D. sinuata seeds (Fig. 5, A and C). The major peak had the same retention time as the TMS derivative of the cis-⌬ 12 isomer of methyl dimorphecolate (9-OH-18:2⌬ 9cis,12cis ). This fatty acid has been previously identified as a very minor component of the seed oil of D. sinuata (27). Although these isomers of dimorphecolic acid were detectable in soybean embryo extracts, the cis-⌬ 12 isomer comprised Ͻ0.1% of the total fatty acids, and dimorphecolic acid (i.e. the trans-⌬ 12 isomer) comprised Ͻ0.05% of the total fatty acids. These results indicated that DsFAD2-2 catalyzes the formation of the C-9 hydroxyl group and the trans-⌬ 10 double bond of dimorphecolic acid. These structural features likely resulted from modification of the ⌬ 9 double bond of lin-oleic acid isomers based on the sequence homology of Ds-FAD2-2 with ⌬ 9 -fatty acid conjugases from C. officinalis. We hypothesized that the cis-⌬ 12 isomer of dimorphecolic acid is formed by DsFAD2-2 activity with cis-⌬ 12 -linoleic acid, the major fatty acid of somatic soybean embryos. Conversely the trans-⌬ 12 double bond of dimorphecolic acid in D. sinuata seeds arises from DsFAD2-2 activity with trans-⌬ 12 -linoleic acid, the product of DsFAD2-1. To test this hypothesis, Ds-FAD2-1 and DsFAD2-2 were co-expressed in soybean somatic embryos. In embryos that produced trans-⌬ 12 -linoleic acid via DsFAD2-1 activity, dimorphecolic acid, rather than its cis-⌬ 12 isomer, accounted for Ͼ90% of the total dimorphecolic acid content (Figs. 4B and 5B). In addition, dimorphecolic acid isomers accounted for ϳ0.5-1.0% of the total fatty acids of the somatic embryos, which was a 5-10-fold increase relative to embryos that expressed DsFAD2-2 alone. These results were consistent with the biosynthesis of dimorphecolic acid by activity of DsFAD2-2 with trans-⌬ 12 -linoleic acid produced by Ds-FAD2-1. Of note, the detection of dimorphecolic acid at amounts of Ͻ0.05% of the fatty acids in embryos that express only DsFAD2-2 (see Fig. 5B) suggests that trace amounts of trans-⌬ 12 -linoleic acid naturally occur in soybean somatic embryos.
The involvement of DsFAD2-1 and DsFAD2-2 in dimorphecolic acid synthesis was examined further by co-expression of these enzymes in S. cerevisiae. This system is particularly advantageous for examining the coordinate activities of Ds-FAD2-1 and DsFAD2-2 because it does not normally produce linoleic acid. Consistent with the results obtained from soybean somatic embryos, co-expression of DsFAD2-1 and DsFAD2-2 in S. cerevisiae was accompanied by the production of dimorphecolic acid (Fig. 6A). This fatty acid was not detected in cells harboring the empty expression vector or in cells transformed with only the DsFAD2-2 cDNA (Fig. 6C). Dimorphecolic acid accounted for ϳ0.5% of the fatty acids of the yeast cells expressing DsFAD2-1 and DsFAD2-2. In addition, a fatty acid with a   mass spectrum consistent with that of 9-OH-16:2⌬ 10trans,12trans was detected in these cells, presumably from the activity of DsFAD2-2 with the 16:2⌬ 9cis,12trans product of DsFAD2-1 (results not shown). The mass spectrum of the TMS-derivatized methyl ester of this fatty acid contained a molecular ion of 354 m/z and an abundant 197 m/z ion from fragmentation between the C-8 and C-9 atoms. It is notable that no 9-OH-18:1⌬ 9cis was detected in cells that expressed only DsFAD2-2. The lack of this product indicates that DsFAD2-2 does not function on oleic acid and that DsFAD2-2 activity occurs after the production of trans-⌬ 12 -linoleic acid by DsFAD2-1.
DsFAD2-2 was also co-expressed in S. cerevisiae with a typical cis-⌬ 12 -oleic acid desaturase from E. lagascae to assess the in vivo activity of this enzyme with the cis-⌬ 12 isomer of linoleic acid. Cells engineered with these enzymes accumulated the cis-⌬ 12 isomer of dimorphecolic acid (Fig. 6B). However, only very small amounts of this fatty acid were formed despite the presence of substantial amounts of the cis-⌬ 12 -linoleic acid substrate produced from the activity of the cis-⌬ 12 -oleic acid desaturase. In these cells, cis-⌬ 12 -dimorphecolic acid accounted for Ͻ0.05% of the total fatty acids, while cis-⌬ 12 -linoleic acid accumulated to ϳ15% of the total fatty acids (data not shown). These results indicate that the cis-⌬ 12 isomer of linoleic acid, in contrast to the trans-⌬ 12 isomer, is a poor substrate for Ds-FAD2-2. This finding is consistent with results obtained from the expression of DsFAD2-2 alone in soybean somatic embryos (Fig. 5A). It is notable that trace amounts of dimorphecolic acid were detected in the yeast cells co-expressing DsFAD2-2 and cis-⌬ 12 -oleic acid desaturase (Fig. 6B). This is similar to the observation from soybean embryos expressing DsFAD2-2 alone (Fig. 5A) and further suggests that the cis-⌬ 12 -oleic acid desaturase displays very low trans-⌬ 12 desaturase activity.
Overall these results indicate that dimorphecolic acid is formed in D. sinuata seeds by the combined activities of Ds-FAD2-1 and DsFAD2-2 through a biosynthetic pathway that involves the formation of trans-⌬ 12 -linoleic acid by DsFAD2-1 activity and the subsequent conversion of the ⌬ 9 double bond of this fatty acid into a C-9 hydroxyl group and trans-⌬ 10 double bond by DsFAD2-2 activity (Fig. 7). FIG. 5. Selected ion chromatograms from GC-MS analyses of TMS-derivatized fatty acid methyl esters from soybean somatic embryos expressing DsFAD2-2 (A), from soybean somatic embryos co-expressing DsFAD2-1 and DsFAD2-2 (B), and from developing D. sinuata seeds (C). Chromatograms were obtained by extracting the 225 m/z ion from total ion chromatograms of TMSderivatized fatty acid methyl esters. This ion is diagnostic for the TMS derivative of methyl dimorphecolate. The cis-⌬ 12 isomer of dimorphecolic acid (9-OH-18:2⌬ 9cis,12cis ) and dimorphecolic acid (9-OH-18: 2⌬ 9cis,12trans ) accounted for Յ0.1% and Յ0.05%, respectively, of the total fatty acids of soybean embryos expressing DsFAD2-2 alone (A). Dimorphecolic acid comprised ϳ1% of the total fatty acids from soybean embryos that co-expressed DsFAD2-1 and DsFAD2-2 (B). The trans-⌬ 12 isomer of linoleic acid, formed by DsFAD2-1 activity, also comprised ϳ10% of the total fatty acids of these embryos.

DISCUSSION
In this study, two divergent forms of FAD2, designated Ds-FAD2-1 and DsFAD2-2, were identified among ESTs from D. sinuata seeds, which accumulate high levels of the unusual fatty acid dimorphecolic acid. DsFAD2-1 was shown to be a trans-⌬ 12 -oleic acid desaturase by expression in yeast and soybean somatic embryos. Using a similar approach, DsFAD2-2 was demonstrated to catalyze the conversion of the ⌬ 9 double bond of linoleic acid isomers into a C-9 hydroxyl group and trans-⌬ 10 double bond. In addition, dimorphecolic acid biosynthesis was conferred to yeast and soybean somatic embryos when DsFAD2-1 and DsFAD2-2 were co-expressed. These results thus provide conclusive evidence that DsFAD2-1 and DsFAD2-2 function in a coordinate manner to produce dimorphecolic acid in D. sinuata seeds. This, to our knowledge, is the first report of the evolution of two divergent FAD2s for the production of an unusual fatty acid in seeds of a single species.
The function of DsFAD2-1 as primarily a trans-⌬ 12 -oleic acid desaturase appears to be novel relative to that of previously characterized members of the FAD2 family. Expression of Ds-FAD2-1 in yeast resulted in the production of trans-⌬ 12 -linoleic acid and only traces amounts of cis-⌬ 12 -linoleic acid. Similarly expression of this enzyme in soybean somatic embryos was accompanied by the accumulation of trans-⌬ 12 -linoleic acid to ϳ15% of the total fatty acids. No other unusual fatty acid products were detected in the transgenic embryos. The ⌬ 12fatty acid conjugase from A. fordii seeds is the only other member of the FAD2 family that has been reported to display trans-⌬ 12 -oleic acid desaturase activity. Dyer et al. (23) have recently shown that small amounts of trans-⌬ 12 -linoleic acid accumulate when this enzyme is expressed in yeast. In contrast to DsFAD2-1, however, trans-⌬ 12 -oleic acid desaturation is a minor activity of the A. fordii ⌬ 12 -fatty acid conjugase in planta. The seed oil of A. fordii contains 80% eleostearic acid from activity of the ⌬ 12 -fatty acid conjugase but Ͻ1% trans-⌬ 12linoleic acid from the alternative activity of this enzyme (23).
DsFAD2-1 and cis-⌬ 12 -oleic acid desaturases likely have very similar catalytic mechanisms but differ in their substrate binding properties. In this regard, the sphingolipid ⌬ 8 -desaturase of plants has been shown to catalyze both cis and trans desaturation of sphingolipid long chain bases (28). It was proposed that the different double bond orientations arise from the stereochemical conformation in which the acyl substrate is presented to the diiron atoms in the active site (18). Based on this proposal, trans-⌬ 12 desaturation of oleic acid occurs when the C-12 and C-13 atoms are presented to the diiron center in a trans conformation, and cis-⌬ 12 occurs when these atoms are presented in the cis conformation. It is therefore likely that DsFAD2-1 has evolved amino acid substitutions that alter the conformation of the fatty acid substrate in the active site relative to cis-⌬ 12 -oleic acid desaturases. Apart from differences in the geometry of substrate binding, removal of hydrogen atoms from acyl chains probably occurs through the same active site chemistry in cis-and trans-⌬ 12 -oleic acid desaturases.
DsFAD2-2 is most closely related to ⌬ 9 -fatty acid conjugases from C. officinalis, which catalyze the synthesis of calendic acid (18:3⌬ 8trans,10trans,12cis ). DsFAD2-2 shares ϳ75% amino acid sequence identity with these enzymes. DsFAD2-2, like the ⌬ 9fatty acid conjugases, modifies the ⌬ 9 double bond of linoleic acid. In addition, both types of enzymes generate a trans-⌬ 10 double bond. The C. officinalis ⌬ 9 -fatty acid conjugases have recently been shown to catalyze the removal of hydrogen atoms from the C-8 and C-11 positions that flank the ⌬ 9 double bond of linoleic acid (8). Removal of a hydrogen atom from the C-11 position appears to be the initial step in the catalytic mechanism of the ⌬ 9 -fatty acid conjugases (8). It is likely that Ds-FAD2-2 operates through a mechanism that is similar to that of the ⌬ 9 -fatty acid conjugases as well as desaturase-related fatty acid hydroxylases (29,30). For example, DsFAD2-2-catalyzed removal of a hydrogen atom from the C-11 position may result in an intermediate that contains a radical on the C-9 atom (Fig. 8). This radical may then remove an oxygen atom from the diiron center because of its close proximity to the catalytic core of DsFAD2-2. The end result of this mechanism would be a C-9 hydroxyl group and a trans-⌬ 10 double bond as found in dimorphecolic acid (Fig. 8). Such a variation on the mechanism of the ⌬ 9 -fatty acid conjugases would be possible if FIG. 7. Proposed biosynthetic pathway of dimorphecolic acid in D. sinuata seeds. The expression studies outlined are consistent with a biosynthetic pathway of dimorphecolic acid involving the trans-⌬ 12 desaturation of oleic acid by the activity of DsFAD2-1 to form trans-⌬ 12 -linoleic acid. The ⌬ 9 double bond of trans-⌬ 12 -linoleic acid is then converted into a 9-OH group and trans-⌬ 10 double bond by the activity of DsFAD2-2 to form dimorphecolic acid. The fatty acid substrates for each reaction are likely bound to a phospholipid (as indicated by R) as has been shown for other FAD2-catalyzed biosynthetic pathways (19).
FIG. 8. Proposed mechanism of DsFAD2-2. The mechanism is based on biochemical studies of the ⌬ 9 -fatty acid conjugase (8) and desaturase-related fatty acid hydroxylases (29,30). As indicated, removal of a C-11 hydrogen atom from trans-⌬ 12 -linoleic acid by the diiron-oxo center of DsFAD2-2 results in the shift of the cis-⌬ 9 double bond to the trans-⌬ 10 position. The radical remaining at the C-9 position is then able to remove an oxygen atom from the diiron-oxo center because of close proximity to the catalytic core of DsFAD2-2 as dictated by the substrate binding properties of this enzyme.
there is a slight difference in the positioning of the fatty acid substrate relative to the diiron center in DsFAD2-2. This difference could arise from relatively small alterations in the conformation of the active site of DsFAD2-2 relative to the ⌬ 9 -fatty acid conjugases. It is notable that the ⌬ 9 -fatty acid conjugases and DsFAD2-2 have slightly different substrate specificities. The ⌬ 9 -fatty acid conjugases are most active with cis-⌬ 12 -linoleic acid (7,8), whereas DsFAD2-2 appears to be most active with trans-⌬ 12 -linoleic acid. For example, soybean somatic embryos that co-express DsFAD2-1 and DsFAD2-2 contain ϳ35% cis-⌬ 12 -linoleic acid and 15% trans-⌬ 12 -linoleic acid. Yet DsFAD2-2 uses trans-⌬ 12 -linoleic acid to the near exclusion of the cis-⌬ 12 isomer for the synthesis of dimorphecolic acid.
The identification of DsFAD2-1 and DsFAD2-2 extends the range of functional outcomes that have been ascribed to the FAD2 family of enzymes. The amino acid sequences of Ds-FAD2-1 and DsFAD2-2 will likely be useful for structure-function studies of FAD2 and other membrane-associated diironoxo enzymes. In addition, the availability of genes for DsFAD2-1 and DsFAD2-2 will facilitate biotechnological studies aimed at producing high levels of dimorphecolic acid in microbes and plants for industrial applications. Our demonstration that dimorphecolic acid biosynthesis can be conferred to yeast and soybean somatic embryos by co-expression of Ds-FAD2-1 and DsFAD2-2 lays the groundwork for such studies.