Formation of Conjugated Delta 8,Delta 10-Double Bonds by Delta 12-Oleic-acid Desaturase-related Enzymes

BIOSYNTHETIC ORIGIN OF CALENDIC ACID*

Edgar B. CahoonDagger, Kevin G. Ripp, Sarah E. Hall, and Anthony J. Kinney

From DuPont Nutrition and Health, Experimental Station, Wilmington, Delaware 19880-0402

Received for publication, October 9, 2000, and in revised form, November 1, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 C18 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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The common polyunsaturated fatty acids of plant seed oils contain cis-double bonds that are separated by a methylene group. The primary examples of such fatty acids are linoleic acid (18:2Delta 9cis,12cis) and alpha -linolenic acid (18:3Delta 9cis,12cis,15cis).1 In contrast, the seed oils of a number of plant species contain polyunsaturated fatty acids with conjugated (or non-methylene-interrupted) double bonds (1). Examples of these unusual fatty acids include alpha -eleostearic acid (18:3Delta 9cis,11trans,13trans), alpha -parinaric acid (18:4Delta 9cis,11trans,13trans,15cis), punicic acid (18:3Delta 9cis,11trans,13cis), and calendic acid (18:3Delta 8trans,10trans,12cis) (1). Seed oils that contain fatty acids with conjugated double bonds display high rates of oxidation compared with oils that contain unsaturated fatty acids with methylene-interrupted double bonds (2). Because of this property, seed oils such as tung oil that are enriched in fatty acids with conjugated double bonds are used commercially as drying agents in paints and varnishes (3).

We have recently demonstrated that the conjugated trans-Delta 11- and trans-Delta 13-double bonds of alpha -eleostearic and alpha -parinaric acids in seeds of Momordica charantia and Impatiens balsamina, respectively, are synthesized by divergent forms of the Delta 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-Delta 12-double bond into conjugated trans-Delta 11- and trans-Delta 13-double bonds (4, 5). This activity contrasts with that of the typical FAD2 desaturase of plants, which introduces a cis-Delta 12-double bond into oleic acid. In M. charantia seeds, alpha -eleostearic acid is formed by modification of the cis-Delta 12-double bond of linoleic acid by a FAD2 conjugase (4). Similarly, the synthesis of alpha -parinaric acid in I. balsamina seeds arises from the conjugase-catalyzed modification of the cis-Delta 12-double bond of alpha -linolenic acid (4). These reactions use fatty acids bound to phosphatidylcholine as substrates (5), as has been shown for other FAD2-type enzymes (6). In addition, based on the mechanism proposed for conjugated double bond synthesis in red algae (7), the production of alpha -eleostearic and alpha -parinaric acids probably involves removal of a hydrogen atom from the C-11 and C-14 methylene groups that flank the cis-Delta 12-double bond of linoleic and alpha -linolenic acids.

Calendic acid, the primary fatty acid of Calendula officinalis seeds (8-10), is a conjugated trienoic fatty acid, like alpha -eleostearic acid, but contains conjugated trans-Delta 8-, trans-Delta 10-, and cis-Delta 12-double bonds. This fatty acid composes 50-60% (w/w) of the seed oil of C. officinalis, but is absent from leaves of this plant (8).2 In common with alpha -eleostearic acid synthesis, linoleic acid has been shown to be the biosynthetic precursor of calendic acid (11, 12). Unlike alpha -eleostearic acid synthesis, however, the conjugated trans-Delta 8- and trans-Delta 10-double bonds of calendic acid arise from modification of the cis-Delta 9-double bond of linoleic acid (11, 12). Based on our previous studies (4) and the proposed mechanism of conjugases (7), it seemed likely that the conjugated trans-Delta 8- and trans-Delta 10-double bonds of calendic acid are formed by a fatty acid desaturase-like enzyme. The involvement of a FAD2-related enzyme in the modification of a cis-Delta 9-double bond, however, has not been previously demonstrated.

In this report, we have undertaken a genomics-based approach to characterize the biosynthetic origin of the conjugated double bonds of calendic acid. By sequencing of random cDNAs derived from developing C. officinalis seeds, we have identified cDNAs for two closely related variant forms of FAD2. Expression of either cDNA in somatic soybean embryos results in the accumulation of calendic acid. These findings thus demonstrate that FAD2-type enzymes can catalyze not only the modification of the Delta 12-position, but also the Delta 9-position of fatty acid substrates. In addition, 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).


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cDNA Library Construction-- Total RNA was isolated from developing seeds of C. officinalis variety Dwarf Gem (Burpee) plants using the method described by Jones et al. (15). Poly(A)+ RNA was enriched from the total RNA and used for cDNA library construction as described previously (4). The resulting library consisted of cDNA inserts cloned directionally (5' to 3') in the EcoRI and XhoI sites of pBluescript II SK(+) and was maintained in Escherichia coli DH10B cells (Life Technologies, Inc.). Bacterial cells harboring the libraries in plasmid form were stored as glycerol stocks at -80 °C until used for expressed sequence tag (EST)3 analysis.

Generation of ESTs and Identification of Divergent FAD2 cDNAs-- Plasmids for EST analysis were prepared from randomly picked colonies from the C. officinalis cDNA library using the QIAGEN REAL Prep 96 system according to the manufacturer's protocol. Nucleotide sequence was obtained from the 5'-ends of cDNAs in pBluescript II SK(+) using the M13 reverse priming site and dye terminator cycle sequencing with an ABI 377 DNA fluorescence sequencer. Partial nucleotide sequences were obtained for 3036 random cDNAs from the C. officinalis developing seed library using this methodology. Putative identities were assigned to these cDNAs by comparison of their partial sequences with translated sequences in the public data bases using the NCBI BLASTX program (16).

From this analysis of the C. officinalis developing seed library, full-length cDNAs for two closely related divergent forms of FAD2 were identified. The polypeptides encoded by these cDNAs were designated CoFADX-1 and CoFADX-2. Nucleotide sequences were determined from both strands of the CoFADX-1 and CoFADX-2 cDNAs in pBluescript II SK(+) by dye terminator sequencing using the instrumentation described above.

Expression of CoFADX-1 and CoFADX-2 cDNAs in Somatic Soybean Embryos-- The vector pKS67 was used for expression of cDNAs for CoFADX-1 and CoFADX-2 in soybean (Glycine max) somatic embryos. This vector contains a unique NotI site for cloning of transgenes that is flanked by the seed-specific promoter of the gene for the alpha '-subunit of beta -conglycinin (17) and phaseolin termination sequence (18). Bacterial selection with this vector is conferred by a hygromycin B phosphotransferase gene (19) under the control of the T7 RNA polymerase promoter, and plant selection is conferred by a second hygromycin B phosphotransferase gene under the control of the cauliflower mosaic virus 35 S promoter.

The coding sequences for CoFADX-1 and CoFADX-2 were amplified by PCR using Pfu polymerase (Stratagene) to generate flanking NotI sites for subcloning into the pKS67 expression vector. Full-length cDNAs for CoFADX-1 and CoFADX-2 were used as templates for PCRs. For amplification of CoFADX-1, the following oligonucleotide primer combination was used: 5'-ttgcggccgcTACACCTAGCTACGTACCATG-3' (sense) and 5'-ttgcggccgTCACGGTACTGATGATGGCAC-3' (antisense). The CoFADX-2 cDNA was amplified using the following primer combination: 5'-agcggccgcTATACCATGGGCAAG-3' (sense) and 5'-tgcggccgcTATGTTAAACTTC-3' (antisense). Note that the sequences shown in lowercase letters contain an added NotI site along with additional bases to facilitate restriction enzyme digestion. The resulting PCR products were subcloned into the intermediate vector pCR-Script AMP SK(+) (Stratagene) according to the manufacturer's protocol. The amplified coding sequence for CoFADX-1 or CoFADX-2 was then released with NotI digestion and subcloned into the corresponding site of the soybean expression vector pKS67.

Gene fusions of the CoFADX-1 and CoFADX-2 cDNAs with the beta -conglycinin promoter and phaseolin termination sequences in vector pKS67 were introduced into soybean embryos of cultivar A2872 or Jack using the particle bombardment method of transformation (4, 20). Selection and propagation of the transgenic somatic soybean embryos have been described previously (4, 20). Expression of CoFADX-1 or CoFADX-2 was confirmed by PCR amplification using sequence-specific primers and first-strand cDNA prepared from total RNA isolated from the transgenic somatic soybean embryos.

Fatty Acid Analysis of Transgenic Soybean Embryos-- Fatty acid methyl esters were prepared from transgenic soybean embryos by transesterification in 1% (w/v) sodium methoxide in methanol. Single soybean embryos were homogenized with a glass stirring rod in 0.5 ml of the sodium methoxide solution and incubated at room temperature for 20 min. At the end of this period, 0.5 ml of 1 M sodium chloride was added, and fatty acid methyl esters were extracted with 0.5 ml of heptane. Fatty acid methyl esters were separated and quantified using a Hewlett-Packard 5890 gas chromatograph fitted with an Omegawax column (30 m × 0.32 mm (inner diameter); Supelco Inc.). The oven temperature was programmed from 220 °C (2-min hold) to 240 °C at a rate of 20 °C/min, and carrier gas was supplied by a Whatman hydrogen generator. Fatty acid methyl esters were also analyzed by GC-MS using a Hewlett-Packard 6890 gas chromatograph interfaced with a Hewlett-Packard 5973 mass selective detector. Samples were separated with an INNOWax column (30 m × 0.25 mm (inner diameter); Hewlett-Packard Co.) or with an HP-5 column (30 m × 0.25 mm (inner diameter); Hewlett-Packard Co.). The oven temperature was programmed from 185 °C (3.5-min hold) to 215 °C (5-min hold) at a rate of 2 °C/min and then to 230 °C at a rate of 5 °C/min. The structures of fatty acid methyl esters with conjugated double bonds were also characterized by GC-MS following Diels-Alder derivatization by reaction with 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) (Aldrich) (21). For these studies, fatty acid methyl esters from transgenic soybean embryos were reacted for 10 s on ice with 0.5 ml of 5 mM MTAD in dichloromethane. The reaction was stopped by the addition of 50 µl of 2,4-hexadiene (Aldrich). The derivatized samples were then dried under nitrogen and resuspended in heptane for GC-MS analysis. MTAD adducts were resolved using either a DB-1 Ht column (15 m × 0.25 mm (inner diameter); J & W Scientific) or an HP-5 column (30 m × 0.25 mm (inner diameter)). The oven temperature was programmed from 185 °C (3-min hold) to 275 °C at a rate of 2.5 °C/min. For identification of calendic acid, the mass spectra of Diels-Alder derivatives prepared from transgenic soybean embryos were compared with those of calendic acid adducts generated from fatty acid methyl esters of C. officinalis seeds.

Expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae-- The activities of CoFADX-1 and CoFADX-2 were characterized by expression of the corresponding full-length cDNAs in S. cerevisiae behind the GAL1 promoter in the vector pESC-URA (Stratagene). The coding sequences of CoFADX-1 and CoFADX-2 were removed as BamHI/XhoI fragments from pBluescript II SK(+) and cloned into the corresponding sites of pESC-URA. The resulting plasmids were introduced into S. cerevisiae INVSc1 cells (Invitrogen) by lithium acetate-mediated transformation (22). Transformed cells were selected for their ability to grow on medium lacking uracil. Individual colonies of transformed cells were then grown for 2 days at 30 °C in medium lacking uracil (0.17% (w/v) yeast nitrogen base without amino acids (Difco), 0.5% (w/v) ammonium sulfate, and 0.18% (w/v) SC-URA (Bio 101, Inc.)) supplemented with glycerol and glucose to final concentrations of 5% (v/v) and 0.5% (w/v), respectively. Cells were then washed twice in the growth medium described above with galactose at a final concentration of 2% (w/v) as the carbon source. The washed cells were then diluted to A600 approx  0.2 in the galactose growth medium that also contained Tergitol type NP-40 (Sigma) at a concentration of 0.2% (w/v). Aliquots of these cells were grown in a volume of 3 ml without exogenous fatty acids or with the addition of oleic acid (18:1Delta 9cis), linoleic acid (18:2Delta 9cis,12cis), or alpha -linolenic acid (18:3Delta 9cis,12cis,15cis) at a final concentration of 0.7 mM. Experiments were also conducted with both linoleic and alpha -linolenic acids added to the medium, each at a concentration of 0.35 mM. Galactose-induced cultures were maintained at 16 °C with shaking (350 rpm). Cells were harvested by centrifugation when cultures reached densities of A600 approx  3-4. Cell pellets from the 3-ml cultures were washed with water and dried under vacuum. The pellets were then resuspended in 0.4 ml of 1% (w/v) sodium methoxide in methanol and incubated at room temperature for 20 min. Fatty acid methyl esters resulting from this direct transesterification of cell pellets were extracted and analyzed by GC and GC-MS as described above for analysis of somatic soybean embryos. Fatty acid methyl esters were also reacted with MTAD, and the resulting Diels-Alder adducts were analyzed by GC-MS as described above.

Northern Blot Analysis-- Total RNA was extracted from leaves and developing seeds of C. officinalis using Trizol (Life Technologies, Inc.) according to the manufacturer's protocol. Total RNA (20 µg) from each tissue and RNA standards were electrophoresed on a 1% (w/v) agarose gel containing formaldehyde. Following electrophoresis, RNA was transferred from the gel to Bright Star-Plus nylon membrane (Ambion Inc.) using NorthernMax transfer buffer (Ambion Inc.). The RNA was fixed to the membrane by UV cross-linking. The membrane was rinsed with 2× SSC and then hybridized with 32P-labeled probes for 18 h at 42 °C in NorthernMax hybridization buffer (Ambion Inc.). Probes were prepared from cDNAs for CoFADX-1 or CoFADX-2 and labeled using random hexamer priming (17). Following incubation with probes, blots were washed for 15 min with 2× SSC and 0.1% SDS at room temperature, then washed for 15 min at room temperature with 0.2× SSC and 0.1% SDS, and finally washed for 15 min at 42 °C with 0.2× SSC and 0.1% SDS. Radioactivity on filters was detected by phosphorimaging. Message sizes were estimated based on mobility relative to a 0.24-9.5 kilobase RNA ladder (Life Technologies, Inc.).

Given the high degree of identity between the open reading frames of the CoFADX-1 and CoFADX-2 cDNAs, probes were prepared primarily from the 3'-untranslated regions of these cDNAs to more specifically distinguish between the expression patterns of the corresponding genes. The probes used for Northern analysis were generated by PCR amplification using Pfu polymerase, and cDNAs for CoFADX-1 and CoFADX-2 were used as templates. PCR products were purified by agarose gel electrophoresis prior to use in labeling reactions. For amplification of the CoFADX-1-specific probe (292 base pairs), the following oligonucleotides were used: 5'-GATTTGAAGTTTCAAATAATC-3' (sense) and 5'-GATAACGCCTTTATTATACTG-3' (antisense). For amplification of the CoFADX-2-specific probe (149 base pairs), the following oligonucleotides were used: 5'-AAAATAAGACTTGAAGTTTAAC-3' (sense) and 5'-GGATAACTCCTTTATTATAC-3' (antisense).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Divergent FAD2 cDNAs in C. officinalis Seeds-- An EST approach was used to determine the biosynthetic origin of calendic acid in C. officinalis seeds. DNA sequences were obtained from the 5'-ends of >3000 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. Based on sequence comparisons, five of these cDNAs corresponded to polypeptides that were more closely related to the "typical" plant FAD2 that is associated with the cis-Delta 12-desaturation of oleic acid. The remaining seven cDNAs were found to encode two closely related polypeptides that were designated CoFADX-1 and CoFADX-2. Of these cDNAs, six encoded CoFADX-1, and one encoded CoFADX-2. The longest full-length cDNAs corresponding to CoFADX-1 and CoFADX-2 contained 1457 and 1295 base pairs, respectively. The amino acid sequences of CoFADX-1 and CoFADX-2 deduced from full-length cDNAs share 94% identity (Fig. 1). These polypeptides, however, share <51% identity with all reported FAD2 and FAD2-like enzymes, including hydroxylases (14, 23), epoxygenases (24), acetylenases (24), and Delta 12-specific conjugases (4) (Figs. 1 and 2).



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Fig. 1.   Comparison of amino acid sequences of divergent forms of FAD2 from C. officinalis (CoFADX-1 and CoFADX-2) with a FAD2 desaturase from Arabidopsis thaliana (AtDes) and FAD2 Delta 12-conjugases from M. charantia and I. balsamina (McConj and IbConj, respectively). Colons indicate amino acids that are identical to those in the CoFADX-1 sequence. Gaps in the alignments are maintained with dashes.



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Fig. 2.   Dendrogram of FAD2 and FAD2-related polypeptides derived from alignment of amino acid sequences using the ClustalX program. The distance along the horizontal axis corresponds to the degree of sequence divergence. The GenBankTM/EBI accession numbers of the amino acid sequences represented in the phylogenetic tree are as follows: Ricinus communis hydroxylase (RcOH), T09839; Arachis hypogaea FAD2 desaturase (AhDES), AAB84262; Glycine max FAD2 desaturase (GmDES), P48630; C. officinalis FAD2 (CoFad2), CAB64256; Crepis palaestina epoxygenase (CpEPOX), CAA76156; Crepis alpina acetylenase (CaACET), CAA76158; CoFADX-1, AF310155; CoFADX-2, AF310156; I. balsamina Delta 12-conjugase (IbCONJ), AAF05915; M. charantia Delta 12-conjugase (McConj), AAF05916; A. thaliana FAD2 desaturase (AtDES), P46313; Lesquerella fendleri hydroxylase (LfOH), AAC32755; C. palaestina FAD2 desaturase (CpDES), CAA76157; Petroselinum crispum FAD2 desaturase (PcDES), T15042; Solanum commersonii FAD2 desaturase (ScDES), T10480; and Helianthus annuus FAD2 desaturase (HaDES), T14269. CoFad2 corresponds to the CoDES sequence described by Fritsche et al. (28).

Using Northern blot analysis, expression of genes for CoFADX-1 and CoFADX-2 was detected in developing seeds, but was not detected in leaves of C. officinalis (Fig. 3). This expression profile is consistent with the seed-specific occurrence of calendic acid in C. officinalis (8).2



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Fig. 3.   Northern blot analysis of the expression of genes for CoFADX-1 and CoFADX-2. Radiolabeled probes derived from cDNAs for CoFADX-1 and CoFADX-2 were hybridized to 20 µg of total RNA isolated from leaves (L) and developing seeds (S) of C. officinalis as shown in A. The ethidium bromide-stained gels corresponding to the blots in A are shown in B. kb, kilobase.

Functional Characterization of Divergent C. officinalis FAD2 Enzymes in Transgenic Plants-- FAD2-related polypeptides are microsomal enzymes that are typically recalcitrant to in vitro assay in solubilized membrane extracts (6). As an alternative method of functional characterization, CoFADX-1 and CoFADX-2 were expressed in somatic soybean embryos to examine their effect on fatty acid content. Like seeds, somatic soybean embryos are rich in triacylglycerols, and the fatty acid composition of transgenic somatic embryos is completely predictive of the fatty acid composition of seeds obtained from regenerated plants (25). In these experiments, expression of cDNAs for CoFADX-1 and CoFADX-2 was placed under the control of the strong seed-specific promoter of the gene for the alpha '-subunit of beta -conglycinin (17). Soybean embryos transformed with expression constructs for either CoFADX-1 or CoFADX-2 were found to accumulate several fatty acids that were not detected in untransformed embryos (Fig. 4). The methyl ester of the most abundant of these fatty acids displayed a gas chromatographic retention time identical to that of the calendic acid methyl ester in extracts from C. officinalis seeds (Fig. 4). In addition, the mass spectrum of this fatty acid methyl ester was identical to that of methyl calendic acid and was characterized by an abundant molecular ion at m/z = 292 (data not shown). To further characterize the identity of this novel fatty acid in soybean embryos transformed with CoFADX-1 or CoFADX-2, fatty acid methyl esters from the transgenic embryos were reacted with MTAD and then analyzed by GC-MS. This reagent readily forms Diels-Alder adducts with conjugated trans,trans-double bonds (21). The product formed from fatty acid methyl esters of the transgenic soybean embryos displayed a mass spectral fragmentation pattern identical to that of the Diels-Alder adduct of calendic acid methyl ester prepared from C. officinalis seeds (Fig. 5). As shown by the mass spectra in Fig. 5, the primary adduct detected resulted from derivatization of the trans-Delta 8- and trans-Delta 10-double bonds of the calendic acid methyl ester, which is consistent with the properties of Diels-Alder reactions (21). These data from transgenic soybean embryos thus demonstrate that CoFADX-1 and CoFADX-2 are associated with the formation of the conjugated trans-Delta 8- and trans-Delta 10-double bonds of calendic acid.



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Fig. 4.   Shown in A and B are the results from gas chromatographic analyses of fatty acid methyl esters prepared from an untransformed somatic soybean embryo and a transgenic embryo expressing CoFADX-2, respectively. Shown in C is a mixture of fatty acid methyl esters from seeds that accumulate different isomers of C18 conjugated trienoic fatty acids. This mixture includes fatty acid methyl esters from seeds of Punica granatum, M. charantia, and C. officinalis. P. granatum seeds accumulate punicic acid (18:3Delta 9cis,11trans,13cis); M. charantia seeds accumulate alpha -eleostearic acid (18:3Delta 9cis,11trans,13trans); and C. officinalis seeds accumulate calendic acid (18:3Delta 8trans,10trans,12cis). Peaks a and b in B were tentatively identified as methyl esters of 18:3Delta 8trans,10trans,12trans and 18:4Delta 8trans,10trans,12cis,15cis, respectively. The labeled peaks correspond to methyl esters of the following fatty acids: 16:0, palmitic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, alpha -linolenic acid; 20:0, eicosanoic acid; and 22:0, docosanoic acid. R.T., retention time.



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Fig. 5.   Mass spectral analysis of Diels-Alder adducts of the methyl ester of calendic acid from C. officinalis seeds (A) and from transgenic somatic soybean embryos expressing CoFADX-2 (B). Adducts were prepared by reaction of fatty acid methyl esters with MTAD, which preferentially reacts with the conjugated trans-Delta 8- and trans-Delta 10-double bonds of methyl calendic acid as shown in A. A similar mass spectrum was obtained from analysis of MTAD derivatives prepared from transgenic soybean embryos expressing CoFADX-1 (data not shown).

Two additional fatty acids (corresponding to peaks a and b in Fig. 4B) were detected in low amounts in the transgenic soybean embryos. The methyl ester of peak a in Fig. 4B displayed a mass spectrum identical to that of the methyl ester of calendic acid (data not shown). However, its gas chromatographic retention time on polar phases was slightly longer than that of methyl calendic acid. Based on these properties, this fatty acid was tentatively identified as the trans-Delta 8,trans-Delta 10,trans-Delta 12-isomer of calendic acid. The mass spectrum of the fatty acid methyl ester corresponding to peak b in Fig. 4B was characterized by a prominent molecular ion at m/z = 290, which is consistent with that of a methyl 18:4 isomer. Based on substrate feeding studies with S. cerevisiae cells expressing CoFADX-1 or CoFADX-2 (described below), this fatty acid was tentatively identified as 18:4Delta 8trans,10trans,12cis,15cis, resulting from the activity of these enzymes with alpha -linolenic acid. These tentatively identified 18:3Delta 8trans,10trans,12trans and 18:4Delta 8trans,10trans,12cis,15cis isomers accounted for <0.5% (w/w) and <0.9% (w/w), respectively, of the total fatty acids of the transgenic somatic soybean embryos. The small amounts of these fatty acids in the transgenic plant tissues limited more detailed characterization of their structures.

In somatic soybean embryos expressing CoFADX-2, calendic acid accumulated to as high as 15-22% (w/w) of the total fatty acids (Table I). Surprisingly, this level of calendic acid accumulation had little effect, if any, on the oleic acid (18:1Delta 9cis) content of the transgenic embryos relative to untransformed controls (Fig. 4 and Table I). This result is in marked contrast to previous reports in which the production of unusual fatty acids from FAD2-like enzymes in transgenic seeds was accompanied by large increases in the relative amounts of oleic acid (4, 13, 14). In addition to calendic acid production, the most notable effect on fatty acid composition of soybean embryos expressing CoFADX-2 was a decrease in linoleic acid content compared with untransformed embryos (Table I). This alteration is consistent with linoleic acid serving as the precursor of calendic acid as described below. Small decreases in palmitic acid content were also observed in transgenic embryos expressing either CoFADX-1 or CoFADX-2 (Table I).


                              
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Table I
Fatty acid composition of somatic soybean embryos from an untransformed line and transgenic lines expressing cDNAs for CoFADX-1 and CoFADX-2
Measurements are presented as weight % of the total fatty acids of embryos and were obtained from independent analyses of four to five single embryos (mean ± S.D.) from each line. ND, not detected.

Substrate Specificities of CoFADX-1 and CoFADX-2 in S. cerevisiae-- CoFADX-1 and CoFADX-2 were expressed in S. cerevisiae to examine the substrate specificities of these enzymes. For these experiments, cDNAs encoding CoFADX-1 and CoFADX-2 were introduced behind the GAL1 promoter in the expression vector pESC-URA. In galactose-induced cells transformed with cDNAs for either polypeptide, calendic acid accumulation was observed only when exogenous linoleic acid was included in the growth medium (Fig. 6A). These results thus confirm that linoleic acid is the precursor of calendic acid via the Delta 9-double bond-modifying activity of CoFADX-1 and CoFADX-2.



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Fig. 6.   Gas chromatographic analyses of fatty acid methyl esters from S. cerevisiae cells expressing CoFADX-1 in medium containing linoleic or alpha -linolenic acid. The gas chromatograms shown in A and C were derived from S. cerevisiae cells containing only the expression vector pESC-URA and grown in medium supplemented with linoleic or alpha -linolenic acid, respectively. The gas chromatograms shown in B and D were derived from cells expressing the CoFADX-1 cDNA in vector pESC-URA and grown in medium containing linoleic or alpha -linolenic acid, respectively. Shown in E are fatty acid methyl esters prepared from C. officinalis seeds. The peak labeled 18:4 in D was tentatively identified as the methyl ester of 18:4Delta 8trans,10trans,12cis,15cis. Peaks labeled with numbers represent methyl esters of the following fatty acids: peak 1, palmitic acid (16:0); peak 2, palmitoleic acid (16:1Delta 9cis); peak 3, stearic acid (18:0); peak 4, oleic acid (18:1Delta 9cis); peak 5, linoleic acid (18:2Delta 9cis,12cis); and peak 6, alpha -linolenic acid (18:3Delta 9cis,12cis,15cis).

Calendic acid accumulation was also dependent on the growth temperature of the cultures, as little or no calendic acid was detected in induced cells maintained at 30 °C. However, the accumulation of this fatty acid was enhanced by reduced growth temperatures (e.g. 16 °C). This temperature dependence of calendic acid accumulation is similar to what has been previously observed with linoleic acid production in S. cerevisiae expressing Arabidopsis FAD2 (26). Calendic acid accounted for as much as 4.5% (w/w) of the total fatty acids of induced S. cerevisiae cells maintained at 16 °C (Table II). Of note, the amount of calendic acid detected in cells expressing CoFADX-1 was at least comparable to that found in cells expressing CoFADX-2 (data not shown).


                              
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Table II
Fatty acid composition of S. cerevisiae cells expressing the cDNA for CoFADX-1 behind the GAL1 promoter in medium supplemented with polyunsaturated fatty acids
For these studies, the CoFADX-1 cDNA was expressed in vector pESC-URA. Separate experiments were conducted with medium containing linoleic acid (+18:2), alpha -linolenic acid (+18:3), or both linoleic and alpha -linolenic acids (+18:2/18:3). The concentration of linolenic and alpha -linolenic acids in the medium was 0.7 mM in the +18:2 and +18:3 treatments. In the +18:2/18:3 experiment, the concentration of each fatty acid in the medium was 0.35 mM. For each experiment, the values shown are the means ± S.D. of the fatty acid compositions of three independent cultures. Of note, no calendic acid or 18:4 was detected in control cultures containing the pESC-URA vector without cDNA insert.

More detailed characterization of substrate specificity was conducted using S. cerevisiae cells expressing CoFADX-1. No conjugated dienoic fatty acids were detected in cells grown without exogenous fatty acid or with added oleic acid (data not shown). These results suggest that CoFADX-1 has no or relatively low activity with oleic acid or with the palmitoleic acid (16:1Delta 9cis) found in high levels in cells not provided with exogenous fatty acids.

The inclusion of alpha -linolenic acid in the medium resulted in the production of a novel fatty acid by cells expressing CoFADX-1 (Fig. 6D). The methyl ester of this fatty acid displayed a retention time identical to that of peak b in Fig. 4B in gas chromatograms of somatic soybean embryos expressing the divergent C. officinalis FAD2 enzymes. In addition, the mass spectrum of this fatty acid methyl ester contained an abundant molecular ion at m/z = 290 (data not shown), which is indicative of an 18:4 isomer. Furthermore, reaction of this novel fatty acid methyl ester with MTAD resulted in the formation of a Diels-Alder adduct (as determined by GC-MS) with a molecular ion at m/z = 403 (data not shown), which is consistent with an 18:4 isomer that contains conjugated double bonds. Given these results and our demonstration that CoFADX-1 and CoFADX-2 convert the cis-Delta 9-double bond of linoleic acid into trans-Delta 8- and trans-Delta 10-double bonds, the 18:4 isomer formed from alpha -linolenic acid (18:3Delta 9cis,12cis,15cis) is probably 18:4Delta 8trans,10trans,12cis,15cis. This fatty acid accounted for ~1.6% (w/w) of the total fatty acids of yeast cells expressing CoFADX-1 in the presence of exogenous alpha -linolenic acid (Table II). The 18:4 isomer was also detected under similar growth conditions in S. cerevisiae cells expressing CoFADX-2 (data not shown).

To examine the relative activity of CoFADX-1 for linoleic and alpha -linolenic acids, cells expressing this enzyme were grown with both fatty acids included in the medium. Although the fatty acids were provided in equal concentrations, alpha -linolenic acid was incorporated by cells to amounts nearly twice that of linoleic acid (46% versus 28% of the total fatty acids) (Table II). Despite this difference, the accumulation of calendic acid (1.8% of the total fatty acids), via activity of CoFADX-l with linoleic acid, was 2-fold greater than the accumulation of 18:4 (0.9% of the total fatty acids), via activity of CoFADX-1 with alpha -linolenic acid (Table II). Similar results were obtained when this experiment was repeated with cells expressing CoFADX-2 (data not shown). These results thus suggest that CoFADX-1 and CoFADX-2 are more active with linoleic acid than with alpha -linolenic acid. This observation is consistent with the higher amounts of calendic acid versus 18:4 that accumulated in the transgenic soybean embryos (Fig. 4B) and in yeast cells expressing CoFADX-1 in the presence of either linoleic acid or alpha -linolenic acid (Table II). Given the recalcitrant nature of FAD2-type enzymes during purification and in vitro assay (6), determination of more detailed kinetic parameters such as Km and Vmax for CoFADX-1 and CoFADX-2 with linoleic and alpha -linolenic acids was not attempted.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have identified cDNAs for two highly expressed FAD2-related polypeptides (CoFADX-1 and CoFADX-2) from C. officinalis seed, a tissue that is enriched in calendic acid (18:3Delta 8trans,10trans,12cis), an unusual conjugated trienoic fatty acid. Expression of cDNAs for CoFADX-1 and CoFADX-2 in soybean somatic embryos or in S. cerevisiae cells was accompanied by the accumulation of calendic acid. Production of this fatty acid in S. cerevisiae cells was dependent on the inclusion of linoleic acid in the medium. These results demonstrate that CoFADX-l and CoFADX-2 are FAD2 conjugases whose expression results in the conversion of the cis-Delta 9-double bond of linoleic acid into trans-Delta 8- and trans-Delta 10-double bonds. This activity thus gives rise to the three conjugated double bonds (trans-Delta 8, trans-Delta 10, and cis-Delta 12) found in calendic acid.

The involvement of FAD2-related enzymes in the modification of the Delta 9-position of fatty acid substrates was unexpected given that all previously described FAD2-type enzymes modify the Delta 12-position of acyl chains. For example, FAD2 desaturase enzymes introduce double bonds 12 carbons from the carboxyl end of substrates that contain a Delta 9-double bond (26, 27). A similar regiospecificity is also displayed by FAD2 hydroxylases from castor (23) and Lesquerella (14). In addition, FAD2-related enzymes modify the cis-Delta 12-double bond of linoleic acid in the synthesis of epoxy groups (24), acetylenic bonds (24), and conjugated Delta 11,Delta 13-double bonds (4). Modification of the cis-Delta 9-double bond of linoleic and alpha -linolenic acids by CoFADX-1 and CoFADX-2 is thus an unprecedented activity among FAD2-related enzymes. Based on the mechanism proposed for conjugated fatty acid synthesis in red algae (7), these enzymes probably function by removal of hydrogen atoms from the Delta 8- and Delta 11-carbons of linoleic and alpha -linolenic acids (28). Given their unique catalytic properties, the primary structures of CoFADX-1 and CoFADX-2 may provide useful comparative information for understanding the structural basis of regiospecificity in FAD2-type enzymes. In this regard, the amino acid sequences of these enzymes contain several insertions and deletions relative to other FAD2-type enzymes (Fig. 1). It is interesting to speculate that these structural features may be associated with the variant regiospecificities of CoFADX-1 and CoFADX-2.

It should be noted that a cDNA for a FAD2-related enzyme (CoDES) from C. officinalis was recently identified by Fritsche et al. (28) and was reported to be a calendic acid-producing desaturase. Note that CoDES is identified as CoFad2 in Fig. 2. However, no gas chromatographic or mass spectral evidence was provided to support this functional identification of CoDES. Interestingly, CoDES shares only 50% amino acid sequence identity with CoFADX-1 and CoFADX-2, but is instead most related to a Crepis FAD2 acetylenase (76% identity) (24). This observation may explain the lack of convincing evidence that calendic acid is produced when this cDNA is expressed in S. cerevisiae (28).2 In addition, we were unable to detect any copies of cDNAs for CoDES in the ~3000 random cDNAs that were sequenced from C. officinalis seeds. In contrast, cDNAs for CoFADX-1 and CoFADX-2 accounted for ~0.23% of the C. officinalis seed ESTs. Of note, we have identified expressed genes encoding FAD2-related polypeptides that share 80-90% amino acid sequence identity with CoDES in a variety of other Asteraceae species, including those that do not accumulate conjugated fatty acids in their seed oils.2 Thus, it seems unlikely that CoDES encodes a seed fatty acid conjugase.

Among the unexpected results from the transgenic production of calendic acid in somatic soybean embryos was the lack of an accompanying high oleic acid phenotype. This finding is in contrast to that previously observed with unusual fatty acid synthesis resulting from the transgenic expression of divergent FAD2 enzymes, including the castor and Lesquerella hydroxylases and the Momordica Delta 12-conjugase (4, 13, 14). In transgenic seeds and somatic soybean embryos that express these enzymes, the accumulation of unusual fatty acids is typically accompanied by 2-4-fold increases in the relative content of oleic acid (4, 13, 14). In contrast to calendic acid, unusual fatty acids such as ricinoleic acid (12-OH-18:1Delta 9cis) and alpha -eleostearic acid (18:3Delta 9cis,11trans,13trans) result from chemical modifications of the Delta 12-position of the C18 fatty acid chain. It is thus possible that unusual fatty acids with modifications of the Delta 12-position directly or indirectly inhibit oleic acid desaturation on phosphatidylcholine in transgenic seeds or embryos. This inhibition apparently does not occur or is more limited with the transgenic production of fatty acids that have similar modifications of the Delta 9-position.

Another unexpected result from the transgenic somatic soybean embryos was the accumulation of small amounts of an 18:4 isomer that we tentatively identified as 18:4Delta 8trans,10trans,12cis,15cis. Based on expression studies with S. cerevisiae, this fatty acid results from the modification of the Delta 9-double bond of alpha -linolenic acid by CoFADX-1 or CoFADX-2 activity. To our knowledge, the occurrence of 18:4 in Calendula seeds has not been previously reported. The lack of detectable 18:4 production is almost certainly due to the fact that alpha -linolenic acid typically composes <1% (w/w) of the total fatty acids of Calendula seeds (Fig. 6E). Therefore, in contrast to somatic soybean embryos, substrate pools of alpha -linolenic acid in Calendula seeds are likely insufficient for 18:4 synthesis.

Amounts of calendic acid in somatic soybean embryos expressing CoFADX-1 and CoFADX-2 were as high as 4% (w/w) and 22% (w/w), respectively, of the total fatty acids of these tissues. Differences in levels of calendic acid accumulation in these lines may be due to factors associated with the transgenic expression of CoFADX-1 and CoFADX-2 or to a lower specific activity of CoFADX-1 in the transgenic soybean embryos. Regardless, amounts of calendic acid resulting from the expression of either CoFADX-1 or CoFADX-2 are likely sufficient to increase the oxidation rate of triacylglycerols in the transgenic soybean embryos. In this regard, seed oils such as tung oil that are enriched in polyunsaturated fatty acids with conjugated double bonds are used as drying agents in coating materials (e.g. paints, varnishes, and inks) because of their high rates of oxidation (2, 3). Therefore, the transgenic expression of CoFADX-1 or CoFADX-2 may ultimately be useful for the production of improved drying oils in existing oilseed crops such as soybean.


    ACKNOWLEDGEMENTS

We thank Dr. Maureen Dolan, Will Krespan, and others of DuPont Genomics for sequencing of cDNA libraries. We also thank Christine Hainey for assistance with cDNA library preparation; Bruce Schweiger, George Cook, and Christine Howells for transforming somatic soybean embryos; Kevin Stecca for providing vector pKS67; and Dr. Brian McGonigle and Rebecca Cahoon for critical reading of the manuscript.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBankTM/EMBL Data Bank with accession number(s) AF310155 (CoFADX-1 cDNA) and AF310156 (CoFADX-2 cDNA).

Dagger To whom correspondence should be addressed: DuPont Experimental Station, E402/4212, Wilmington, DE 19880-0402. Tel.: 302-695-4348; Fax: 302-695-8480; E-mail: Edgar.B.Cahoon@usa.dupont.com.

Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M009188200

1 The fatty acid nomenclature used in this work is as follows. X:Y indicates that the fatty acid contains X numbers of carbon atoms and Y numbers of double bonds. Delta z indicates that a double bond is located at the zth carbon atom relative to the carboxyl end of the fatty acid.

2 E. B. Cahoon, K. G. Ripp, S. E. Hall, and A. J. Kinney, unpublished results.


    ABBREVIATIONS

The abbreviations used are: EST, expressed sequence tag; PCR, polymerase chain reaction; GC-MS, gas chromatography-mass spectrometry; MTAD, 4-methyl-1,2,4-triazoline-3,5-dione.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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