Dimorphecolic Acid Is Synthesized by the Coordinate Activities of Two Divergent Δ12-Oleic Acid Desaturases
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m314329200
ISSN1083-351X
AutoresEdgar B. Cahoon, Anthony J. Kinney,
Tópico(s)Nitrogen and Sulfur Effects on Brassica
ResumoDimorphecolic 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-18:2Δ10trans,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-18:2Δ10trans,12trans) 1Fatty acid nomenclature: X:Y indicates that the fatty acid contains X number of carbon atoms and Y number of double bonds. Δz indicates that a double bond is located at the zth carbon atom relative to the carboxyl end of the fatty acid. is an unusual C18 fatty acid that can comprise more than 60% of the seed oil of Dimorphotheca species (1Smith Jr., C.R. Wilson T.L. Melvin E.H. Wolff I.A. J. Am. Oil Chem. Soc. 1960; 82: 1417-1421Crossref Scopus (57) Google Scholar). This fatty acid has received attention because of its potential value for industrial applications. For example, the chemical functionalities resulting from its C-9 hydroxyl group and conjugated Δ10,Δ12 double bonds make dimorphecolic acid useful in the manufacture of paints, inks, lubricants, plastics, and nylon (2Hof L. Janick J. Progress in New Crops. American Society of Horticultural Science Press, Arlington, VA1996: 372-377Google Scholar, 3Muuse B.G. Cuperus F.P. Derksen J.T.P. Ind. Crops Prod. 1992; 1: 57-65Crossref Scopus (69) Google Scholar). 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 Δ10,Δ12 double bonds, and (iii) a trans-Δ12 double bond. With regard to the trans-Δ12 double bond, Morris and Marshall (4Morris L.J. Marshall M.O. Chem. Ind. August 27. 1966; : 1493-1494Google Scholar) reported nearly 40 years ago that the unusual Δ9cis,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 18:2Δ9cis,12trans is the precursor of dimorphecolic acid rather than the more commonly occurring 18:2Δ9cis,12cis. This initial step in the synthesis of dimorphecolic acid would therefore require an unusual enzymatic activity that generates a trans-Δ12 double bond instead of the cis-Δ12 double bond that is normally formed by the Δ12-oleic acid desaturase (FAD2) 2The abbreviations used are: FAD2, Δ12-oleic acid desaturase; EST, expressed sequence tag; TMS, trimethylsilyl; GC, gas chromatography; MS, mass spectrometry. in plants. When considering the possible biosynthetic origin of the C-9 hydroxyl group and the Δ10 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 (18:3Δ8trans,10trans,12cis), an unusual conjugated fatty acid that has some structural similarity to dimorphecolic acid (5McClean J. Clark A.H. J. Chem. Soc. 1956; 1956: 777-778Google Scholar). It has been previously shown that calendic acid is formed by the conversion of Δ9 double bond of linoleic acid to conjugated Δ8,Δ10 double bonds by the activity of a divergent form of FAD2 (6Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 7Qiu X. Reed D.W. Hong H. MacKensie S.L. Covello P.S. Plant Physiol. 2001; 125: 847-855Crossref PubMed Scopus (47) Google Scholar). This enzyme, which has been termed a "Δ9-fatty acid conjugase," is believed to catalyze the removal of hydrogen atoms from the C-8 and C-11 atoms that flank the Δ9 double bond of linoleic acid (8Reed D.W. Savile C.K. Qiu X. Buist P.H. Covello P.S. Eur. J. Biochem. 2002; 269: 5024-5029Crossref PubMed Scopus (24) Google Scholar). Given the close taxonomic relation of the Calendula and Dimorphotheca genera, it can be speculated that the C-9 hydroxyl group and trans-Δ10 double bond of dimorphecolic acid arise from modification of the Δ9 double bond of 18:2Δ9cis,12trans by the activity of an enzyme that is structurally and functionally related to the Calendula Δ9-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-Δ12 double bond of 18:2Δ9cis,12trans and (ii) an enzyme that subsequently converts the Δ9 double bond of 18:2Δ9cis,12trans into a C-9 hydroxyl group and Δ10trans 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 Δ9- and Δ12-fatty acid conjugases (6Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 9Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (169) Google Scholar), cyclopropane fatty-acid synthase (10Bao X. Katz S. Pollard M. Ohlrogge J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7172-7177Crossref PubMed Scopus (121) Google Scholar), and a cytochrome P450 Δ12-expoxygenase (11Cahoon E.B. Ripp K.G. Hall S.E. McGonigle B. Plant Physiol. 2002; 128: 615-624Crossref PubMed Scopus (100) Google Scholar). 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. 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. (12Jones A. Davies H.M. Voelker T.A. Plant Cell. 1995; 7: 359-371Crossref PubMed Scopus (249) Google Scholar). Poly(A)+ RNA enrichment and cDNA library construction were performed as described previously (6Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 9Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (169) Google Scholar). The cDNA inserts were cloned directionally in the EcoRI and XhoI sites of pBluescript II SK(+). 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 5′ ends of the resulting plasmid as described previously (6Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 9Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (169) Google Scholar). Tentative identification of polypeptides corresponding to the sequenced cDNAs was determined by bioinformatic analysis of translated 5′ 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™ 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 (13Geitz R.D. Woods R.A. Johnston J.A. Molecular Genetics of Yeast: Practical Approaches. Oxford University Press, New York1994: 121-134Google Scholar). 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 A600 ≈ 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 A600 ≈ 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-Δ12-oleic acid desaturase from Euphorbia lagascae (GenBank™ 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: 5′-TATGCGGCCGCAAATGGGAGCAGGAGGTTG-3′ (sense) and 5′-TTTGCGGCCGCATTACATCTTATTCTTGTACC-3′ (antisense). (Note that the underlined sequences correspond to the added NotI restriction sites.) The product was subcloned into the vector pCR-Script AMP SK(+) (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-Δ12-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 α′-subunit of β-conglycinin gene in the vector pKS67 (9Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (169) Google Scholar). 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 5′-TGCGGCCGCAATGGGTGGAGGGATGGGAGCATCTGAG-3′ (sense) and 5′-TAGCGGCCGCTGATTAATCAAGTCTTAG-3′ (antisense). The PCR products were subcloned into the intermediate vector pCR-Script AMP SK(+) (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 DsFAD2-1-derived NotI fragment was linked to the promoter of the gene for the α′-subunit of β-conglycinin in the previously described vector pKS17 (14Cahoon E.B. Marillia E.-F. Stecca K.L. Hall S.E. Taylor D.C. Kinney A.J. Plant Physiol. 2000; 124: 243-251Crossref PubMed Scopus (91) Google Scholar). 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 β-conglycinin promoter and phaseolin 3′ non-translated region in vector pKS67 were introduced into soybean somatic embryos by using particle bombardment (15Finer J.J. McMullen M.D. In Vitro Cell Dev. Biol. 1991; 27: 175-182Crossref Scopus (215) Google Scholar). 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 DsFAD2-2-containing 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 (14Cahoon E.B. Marillia E.-F. Stecca K.L. Hall S.E. Taylor D.C. Kinney A.J. Plant Physiol. 2000; 124: 243-251Crossref PubMed Scopus (91) Google Scholar). 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 (6Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 9Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (169) Google Scholar). 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 (6Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). 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 × 0.25-mm inner diameter, 0.25-μm 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 18:2Δ9,12 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 (16Nilsson R. Liljenberg C. Phytochem. Anal. 1991; 2: 253-259Crossref Scopus (38) Google Scholar). 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 (6Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 9Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (169) Google Scholar). The recovered fatty acid methyl esters were analyzed by GC with an Omegawax 320 column (30-m × 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 °Cat 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 μl 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 (11Cahoon E.B. Ripp K.G. Hall S.E. McGonigle B. Plant Physiol. 2002; 128: 615-624Crossref PubMed Scopus (100) Google Scholar). 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 Δ12 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 (17Agilent Technologies Chromatography Supplies and Reference Guide 2002–2003. Agilent Technologies, Wilmington, DE2002: 391-392Google Scholar) 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 (17Agilent Technologies Chromatography Supplies and Reference Guide 2002–2003. Agilent Technologies, Wilmington, DE2002: 391-392Google Scholar) as was observed in the analysis of soybean extracts (see Fig. 3).Fig. 3Gas chromatographic analyses of fatty acid methyl esters prepared from non-transformed soybean somatic embryos (A), soybean somatic embryos expressing DsFAD2-1 (B), and developing D. sinuata seeds (C). Fatty acid methyl esters were resolved on an OmegaWax 320 GC column as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (18Sperling P. Trernes P. Zank T.K. Heinz E. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 68: 73-95Abstract Full Text Full Text PDF Scopus (241) Google Scholar). 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-Δ12-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 (18Sperling P. Trernes P. Zank T.K. Heinz E. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 68: 73-95Abstract Full Text Full Text PDF Scopus (241) Google Scholar, 19Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (731) Google Scholar). The consensus sequence of the first His box is HECGHH. In all members of the FAD2 family that function as cis-Δ12-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 (20van de Loo F.J. Broun P. Turner S. Somerville C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6743-6747Crossref PubMed Scopus (311) Google Scholar, 21Broun P. Boddupalli S. Somerville C. Plant J. 1998; 13: 201-210Crossref PubMed Scopus (140) Google Scholar, 22Lee M. Lenman M. Banas A. Bafor M. Singh S. Schweizer M. Nilsson R. Liljenberg C. Dahlqvist A. Gummeson P.O. Sjodahl S. Green A. Stymne S. Science. 1998; 280: 915-918Crossref PubMed Scopus (208) Google Scholar). 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-Δ12-oleic acid desaturases. The other four D. sinuata ESTs identified as cis-Δ12-oleic acid desaturases had Ala preceding the first His box and other sequence elements consistent with the cis-Δ12-oleic acid desaturase functional class of FAD2 enzymes (23Dyer J.M. Chapital D.C. Kuan J.-C.W. Mullen R.T. Turner C. McKeon T.A. Pepperman A.B. Plant Physiol. 2002; 130: 2027-2038Crossref PubMed Scopus (148) Google Scholar). Interestingly the amino acid sequences of DsFAD2-1 and DsFAD2-2 share only 48% identity. DsFAD2-1 is most closely related to a cis-Δ12-oleic acid desaturase from Helianthus annuus and shares ∼60–75% amino acid sequence identity with all known cis-Δ12-oleic acid desaturases. This polypeptide also shares 63–67% identity with FAD2-type fatty acid hydroxylases and Δ12-fatty acid conjugases from Aleurites fordii (23Dyer J.M. Chapital D.C. Kuan J.-C.W. Mullen R.T. Turner C. McKeon T.A. Pepperman A.B. Plant Physiol. 2002; 130: 2027-2038Crossref PubMed Scopus (148) Google Scholar) and Punica granatum (24Hornung E. Pernstich C. Feussner I. Eur. J. Biochem. 2002; 269: 4852-4859Crossref PubMed Scopus (81) Google Scholar) but shares ≤55% identity with FAD2 epoxygenases, acetylenases, and Δ9-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 ≤55% identity with all members of the FAD2 family except the Δ9-fatty acid conjugases from Calendula officinalis (6Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 7Qiu X. Reed D.W. Hong H. MacKensie S.L. Covello P.S. Plant Physiol. 2001; 125: 847-855Crossref PubMed Scopus (47) Google Scholar). DsFAD2-2 and the C. officinalis Δ9-fatty acid conjugases share ∼75% amino acid sequence identity, and these polypeptides display a close phylogenetic relationship (Fig. 1). The Δ9-fatty acid conjugases catalyze the conversion of the cis-Δ9 double bond of linoleic acid into trans-Δ8 and trans-Δ10 double bonds. The close relationship of the primary structures of Ds-FAD2-2 and the C. officinalis Δ9-fatty acid conjugases suggested that these enzymes have related functional properties. Functional Characterization of DsFAD2-1—Enzymes of the FAD2 family have typically proven recalcitrant to direct assay (19Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (731) Google Scholar). 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 (6Cahoon E.B. Ripp K.G. Hall S.E. Kinney A.J. J. Biol. Chem. 2001; 276: 2637-2643Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 7Qiu X. Reed D.W. Hong H. MacKensie S.L. Covello P.S. Plant Physiol. 2001; 125: 847-855Crossref PubMed Scopus (47) Google Scholar, 23Dyer J.M. Chapital D.C. Kuan J.-C.W. Mullen R.T. Turner C. McKeon T.A. Pepperman A.B. Plant Physiol. 2002; 130: 2027-2038Crossref PubMed Scopus (148) Google Scholar, 24Hornung E. Pernstich C. Feussner I. Eur. J. Biochem. 2002; 269: 4852-4859Crossref PubMed Scopus (81) Google Scholar, 25Covello P.S. Reed D.W. Plant Physiol. 1996; 111: 223-226Crossref PubMed Scopus (104) Google Scholar). In addition, we have previously used soybean somatic embryos for analysis of FAD2 enzymes (9Cahoon E.B. Carlson T.J. Ripp K.G. Schweiger B.J. Cook G.A. Hall S.E. Kinney A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12935-12940Crossref PubMed Scopus (169) Google Scholar, 11Cahoon E.B. Ripp K.G. H
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