Δ12-Oleate Desaturase-related Enzymes Associated with Formation of Conjugated trans-Δ11, cis-Δ13 Double Bonds
2003; Elsevier BV; Volume: 278; Issue: 7 Linguagem: Inglês
10.1074/jbc.m210748200
ISSN1083-351X
AutoresM. Iwabuchi, Junko Kohno‐Murase, Jun Imamura,
Tópico(s)Nitrogen and Sulfur Effects on Brassica
ResumoConjugated linolenic acids are present as major seed oils in several plant species. Punicic acid (or trichosanic acid) is a conjugated linolenic acid isomer containingcis-Δ9, trans-Δ11, cis-Δ13 double bonds in the C18 carbon chain. Here we report cDNAs, TkFac and PgFac, isolated from Trichosanthes kirilowii and Punica granatum, that encode a class of conjugases associated with the formation of trans-Δ11,cis-Δ13 double bonds. Expression of TkFac and PgFac inArabidopsis seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid up to ∼10% (w/w) of the total seed oils. In contrast, no punicic acid was found in lipids from leaves even when the conjugases were driven under control of the cauliflower mosaic virus 35S promoter. In yeast cells grown without exogenous fatty acids in the culture medium, TkFac and PgFac expression resulted in punicic acid accumulation accompanied by 16:2Δ9cis, 12cisand 18:2Δ9cis, 12cis production. Thus, TkFac and PgFac are defined as bifunctional enzymes having both conjugase and Δ12-oleate desaturase activity. Furthermore, we demonstrate that 16:2Δ9cis, 12cis and 18:3Δ9cis, 12cis, 15cisas well as 18:2Δ9cis, 12cis are potential substrates for the conjugases to form trans-Δ11 and cis-Δ13 double bonds. Conjugated linolenic acids are present as major seed oils in several plant species. Punicic acid (or trichosanic acid) is a conjugated linolenic acid isomer containingcis-Δ9, trans-Δ11, cis-Δ13 double bonds in the C18 carbon chain. Here we report cDNAs, TkFac and PgFac, isolated from Trichosanthes kirilowii and Punica granatum, that encode a class of conjugases associated with the formation of trans-Δ11,cis-Δ13 double bonds. Expression of TkFac and PgFac inArabidopsis seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid up to ∼10% (w/w) of the total seed oils. In contrast, no punicic acid was found in lipids from leaves even when the conjugases were driven under control of the cauliflower mosaic virus 35S promoter. In yeast cells grown without exogenous fatty acids in the culture medium, TkFac and PgFac expression resulted in punicic acid accumulation accompanied by 16:2Δ9cis, 12cisand 18:2Δ9cis, 12cis production. Thus, TkFac and PgFac are defined as bifunctional enzymes having both conjugase and Δ12-oleate desaturase activity. Furthermore, we demonstrate that 16:2Δ9cis, 12cis and 18:3Δ9cis, 12cis, 15cisas well as 18:2Δ9cis, 12cis are potential substrates for the conjugases to form trans-Δ11 and cis-Δ13 double bonds. A large number of fatty acid species have been found in plant seed oils. Typically, plant seeds contain saturated and unsaturated fatty acids, such as palmitic (16:0), palmitoleic (16:1Δ9cis), stearic (18:0), oleic (18:1Δ9cis), linoleic (18:2Δ9cis, 12cis), and α-linolenic (18:3Δ9cis, 12cis, 15cis) acids. These are typical fatty acids with all other fatty acids regarded as unusual. Typically, polyunsaturation of fatty acids is methylene (-CH2-)-interrupted and occurs incis-configuration as found in linoleic and linolenic acids. In contrast, conjugated (non-methylene-interrupted) fatty acids contain double bonds in cis- or trans-configuration. The conjugated fatty acids occur as diene, triene, and tetraene in which the most common conjugated polyenoic acids are octadecatrienoic acids, termed CLNAs. 1The abbreviations used are: CLNA, conjugated linolenic acid; CaMV, cauliflower mosaic virus; FAD2, Δ12-oleate desaturase; GC, gas chromatography; MS, mass spectrometry; FAME, fatty acid methyl ester; CLA, conjugated linoleic acid 1The abbreviations used are: CLNA, conjugated linolenic acid; CaMV, cauliflower mosaic virus; FAD2, Δ12-oleate desaturase; GC, gas chromatography; MS, mass spectrometry; FAME, fatty acid methyl ester; CLA, conjugated linoleic acid Positional and geometrical isomers of CLNA, three 8,10,12-trienes and four 9,11,13-trienes, have been reported to occur naturally (1Smith Jr., C.R. Prog. Chem. Fats Other Lipids. 1970; 11: 137-177Crossref Scopus (99) Google Scholar). Five CLNA isomers occur as major seed oils of several plants: α-eleostearic (cis-Δ9, trans-Δ11, trans-Δ13), calendic (trans-Δ8, trans-Δ10,cis-Δ12), punicic (cis-Δ9,trans-Δ11, cis-Δ13), jacaric (cis-Δ8, trans-Δ10, cis-Δ12), and catalpic (trans-Δ9, trans-Δ11,cis-Δ13) acids (1Smith Jr., C.R. Prog. Chem. Fats Other Lipids. 1970; 11: 137-177Crossref Scopus (99) Google Scholar). These isomers have closely related structure; for example, α-eleostearic and jacaric acids are geometrical isomers of punicic and calendic acids, respectively. CLNAs are major seed oils in plants such as tung (Aleurites fordii), karela (Momordica charantia), marigold (Calendula officinalis), and pomegranate (Punica granatum). Tung oil contains high levels of 9,11,13-triene (α-eleostearic acid) and is used mainly in quick-drying enamels and varnishes. There also is growing evidence showing that supplementation with CLNA has cytotoxic effects on tumor cells and that uptake of CLNA has an effect on lipid metabolism (2Igarashi M. Miyazawa T. Cancer Lett. 2000; 148: 173-179Crossref PubMed Scopus (153) Google Scholar, 3Suzuki R. Noguchi R. Ota T. Abe M. Miyashita K. Kawada T. J. Am. Oil Chem. Soc. 2001; 36: 477-482Google Scholar, 4Koba K. Akahoshi A. Yamasaki M. Tanaka K. Yamada K. Iwata T. Kamegai T. Tsutsumi K. Sugano M. J. Am. Oil Chem. Soc. 2002; 37: 343-350Google Scholar). Conjugated eicosapentaenoic and docosahexaenoic acids with conjugated trienoic structure also exhibit cytotoxic effects on tumor cells (5Igarashi M. Miyazawa T. Biochem. Biophys. Res. Commun. 2000; 270: 649-656Crossref PubMed Scopus (70) Google Scholar). It has been further suggested that the biological action of each conjugated fatty acid may not be equivalent (3Suzuki R. Noguchi R. Ota T. Abe M. Miyashita K. Kawada T. J. Am. Oil Chem. Soc. 2001; 36: 477-482Google Scholar). Previous studies have indicated that linoleic acid is the acyl precursor of α-eleostearic acid and linoleoyl phosphatidylcholine is the precursor of α-eleostearoyl phosphatidylcholine (6Liu L. Hammond G. Nikolau B.J. Plant Physiol. 1997; 113: 1343-1349Crossref PubMed Scopus (28) Google Scholar). Recently cDNAs encoding enzymes that catalyze the formation of the conjugated double bonds in CLNA have been identified. These enzymes were termed conjugases and were shown to be divergent forms of Δ12-oleate desaturase (FAD2). Two types of conjugases associated with the formation of conjugated double bonds intrans-configuration have been identified: one catalyzes the conversion of a cis-Δ12 double bond into the conjugatedtrans-Δ11, trans-Δ13 double bonds found in α-eleostearic acid (7Cahoon 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 (163) Google Scholar), and the other modifies a cis-Δ9 double bond into the trans-Δ8, trans-Δ10 double bonds of calendic acid (8Cahoon 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 (95) Google Scholar, 9Qiu X. Reed D.W. Hong H. MacKenzie S.L. Covello P.S. Plant Physiol. 2001; 125: 847-855Crossref PubMed Scopus (42) Google Scholar). Very recently a class of conjugase associated with the formation of cis-Δ11,trans-Δ13 double bonds of punicic acid was identified (10Hornung E. Pernstich C. Feussner I. Eur. J. Biochem. 2002; 269: 4852-4859Crossref PubMed Scopus (78) Google Scholar). Although the primary structures of these conjugases are similar, each enzyme specifically catalyzes the formation of α-eleostearic, punicic, or calendic acids. Other classes of conjugases associated with the formation of conjugated double bonds in jacaric and catalpic acids have not been reported. It is known that Trichosanthes kirilowii and P. granatum accumulate punicic acid specifically in seeds up to ∼40 and ∼80% (w/w) of the total seed oil, respectively (11Joh Y.-G. Kim S.-J. Christie W. J. Am. Oil Chem. Soc. 1995; 72: 1037-1042Crossref Scopus (37) Google Scholar, 12Takagi T. Itabashi Y. Lipids. 1981; 16: 546-551Crossref Scopus (100) Google Scholar). The biochemical process resulting in high punicic acid accumulation in the seeds of these plants, however, has not been clear. In this study, for the first step in understanding CLNA accumulation in plant seed oils, we isolated cDNAs that encode enzymes associated with the formation of punicic acid from T. kirilowii andP. granatum and expressed them in Arabidopsisplants. We also analyzed the function of these enzymes in yeast cells. Interestingly the enzymes were demonstrated to possess both Δ12-oleate desaturase and conjugase activities. The finding supports the idea that conjugases have diverged from Δ12-oleate desaturase. The growing number of identified primary structures of fatty acid-modifying enzymes provides valuable information to understand the functional divergence of these enzymes. Total RNA was isolated from maturing seeds of T. kirilowii and P. granatum by the methods of Carpenter et al. (13Carpenter C.D. Simon A. Methods Mol. Biol. 1998; 82: 85-89PubMed Google Scholar). The first strand cDNA was synthesized with an oligo-dT primer and Superscript II reverse transcriptase (Invitrogen) from the total RNA and used for PCR amplification. To isolate cDNA fragments, degenerate primers were designed to target conserved amino acid sequences in FAD2-related enzymes. For T. kirilowii, a set of degenerate primers, 5′-TGYGGNCAYCAYGCNTTYAGYGAYTAYCART-3′ (forward primer) and 5′-GGRTGNGTRTGYTGNARNKMNGT-3′ (reverse primer) were used to target the amino acid sequences CGHHAFSDYQ and T(Y/A)LQHTHP, respectively. For P. granatum, 5′-TGYGGNCAYMRNGCNTTYWSNGAYTAYCAR-3′ (forward primer) and 5′-KYNCCNCKNARCCARTYCCAYTC-3′ (reverse primer) were used to target the amino acids CGH(H/R)AFSDYQ and EW(D/N)WLRG(A/N), respectively. PCR amplification was performed with TaKaRa Ex Taq (Takara Shuzo) with 30 cycles of 30 s at 94 °C, 1 min at 50 °C, and 40 s at 72 °C followed by an extension step for 10 min at 72 °C. The amplified products (∼0.5 kb) were cloned into pGEM-T Easy plasmid vector (Promega) and sequenced using a PRISM DyeDeoxy Terminator Cycle Sequencing System (Applied Biosystems). The sequence analysis revealed that two types of cDNA fragments closely related to FAD2 were isolated in each experiment forT. kirilowii (TkFac and TkFad2) and P. granatum (PgFac and PgFad2). The second strand cDNA was synthesized with a Marathon cDNA Amplification kit (Clontech). Adaptor ligation to the double strand cDNAs and 5′- and 3′-rapid amplification of cDNA ends was performed according to the manufacturer's protocol. cDNA fragments containing 5′ and 3′ regions were cloned into pGEM-T Easy and sequenced. Finally, full-length cDNAs of four FAD2-related cDNAs were isolated by PCR amplification with Pyrobest DNA polymerase (Takara Shuzo) using a set of primers corresponding to the sequences in 5′- and 3′-untranslated regions. The PCR amplification consisted of 25 cycles of 30 s at 94 °C, 1 min at 55 °C, and 2.5 min at 72 °C followed by an extension step for 10 min at 72 °C. The PCR product was incubated with TaKaRa Ex TaqDNA polymerase (Takara Shuzo) for 10 min at 72 °C. The PCR products from four independent amplifications for a set of primers were cloned into pGEM-T Easy and sequenced as described above. The coding regions of TkFac and PgFac were amplified by PCR using Pyrobest DNA polymerase. For amplification of TkFac, the forward primer was designed with a flanking BamHI site (5′-taggatccATGGGAGGTTGGTGAAGGAATAG-3′), and the reverse primer corresponded to the sequence 5–27 bp downstream from the stop codon of TkFac with a flanking SacI site (5′-atgagctcGATGATATCATGAAACCAAGAGG-3′). For amplification of PgFac, the forward primer was designed with flankingEcoRV/XbaI sites (5′-tagatatctagaATGGGAGCTGATGGAACAATGTCTC-3′), and the reverse primer corresponded to the sequence 7–33 bp downstream from the stop codon of PgFac with a flanking SacI site (5′-atgagctcGATATTAGGTTCGATTCTAATAAC-3′). The PCR amplification and cloning into pGEM-T Easy were as described above. pGEM-T Easy/TkFac was digested with BamHI and SacI and ligated at theBamHI/SacI site of the pGEM-3Z plasmid vector (Promega). To generate plant expression vectors with the CaMV 35S promoter and nos terminator (pKS-TkFac and pKS-PgFac), the cDNA sequences were released from pGEM-3Z/TkFac and from pGEM-T Easy/PgFac with XbaHI/SacI digestion and then cloned into the corresponding sites of a binary vector pLAN421 (14Uematsu C. Murase M. Ichikawa H. Imamura J. Plant Cell Rep. 1991; 10: 286-290Crossref PubMed Scopus (48) Google Scholar) in which theGUS gene has been eliminated withXbaI/SacI digestion. To generate plant expression vectors with a strong seed-specific promoter of theBrassica napus napin gene (15Josefsson L.-G. Lenman M. Ericson M.L. Rask L. J. Biol. Chem. 1987; 262: 12196-12201Abstract Full Text PDF PubMed Google Scholar) (pKN-TkFac and pKN-PgFac), the XbaI-SacI fragments were cloned into binary vector pNGKM (16Kohno-Murase J. Murase M. Ichikawa H. Imamura J. Plant Mol. Biol. 1994; 26: 1115-1124Crossref PubMed Scopus (49) Google Scholar) in which the GUS gene had been eliminated with XbaI/SacI digestion. A. thaliana ecotype Columbia (Col-0) was transformed with the conjugase construct by vacuum infiltration methods (17Bechtold N. Ellis J. Pelletier G. Life Sci. 1993; 316: 1194-1199Google Scholar). Five plants per 20-cm2 pot were raised at 21 °C under fluorescent illumination with a 16-h light/8-h dark cycle.Agrobacterium strain EHA101 (pEHA) (18Hood E.E. Helmer G.L. Fraley R.T. Chilton M.-D. J. Bacteriol. 1986; 168: 1291-1301Crossref PubMed Scopus (754) Google Scholar) harboring the conjugase construct was cultured overnight at 30 °C in YEB medium (0.1% yeast extract, 0.5% beef extract, 0.5% peptone, and 0.5% sucrose, pH 7.0) supplemented with 50 μg/ml kanamycin, 25 μg/ml chloramphenicol, 100 μg/ml spectinomycin, and 2.5 μg/ml tetracycline the day before infiltration. Plants were dipped withAgrobacterium suspension diluted toA 600 = 0.8 in dipping solution containing 0.02% Silwet L-77 and 0.044 μm 6-benzylaminopurine for 15 min under vacuum and then placed in a covered tray. The next day the plants were uncovered, set upright, and allowed to grow for ∼4 weeks in a growth chamber under the conditions described above. When the siliques were matured, seeds were harvested and planted for selection of positive transformants. Seeds were surface-sterilized by immersion for 2 min in 70% ethanol followed by 15 min in 5% bleach solution containing 1% SDS and then rinsed five times with sterile water. Transformants were selected for by survival on Murashige-Skoog medium containing 30 μg/ml kanamycin and 250 μg/ml carbenicillin. The BamHI-SacI fragment of TkFac cDNA released from pGEM-3Z/TkFac as described above was cloned into the yeast expression vector pYES2 with the galactose-inducibleGAL1 promoter (Invitrogen) to generate pYES2/TkFac. pGEM-T Easy/PgFac was digested with EcoRV/SacI and cloned into the plasmid vector pBluescript II (Stratagene), and then the PgFac cDNA was released withHindIII/SacI digestion and cloned into pYES2 to generate pYES2/PgFac. The coding regions of TkFad2 and PgFad2 were amplified by PCR using Pyrobest DNA polymerase and cloned into pGEM-T Easy as described above. For amplification of TkFad2, the forward primer was designed with a flanking SmaI site (5′-atcccgggATGGAAAAGGGCGTTCAAGAGC-3′), and the reverse primer corresponded to the sequence 1–24 bp downstream from the stop codon of TkFad2 with a flanking SacI site (5′-atgagctcCAATTTTGACTCAAGACAGAC-3′). For amplification of PgFad2, the forward primer was designed with flankingHindIII/XbaI sites (5′-taaagctctagaATGGGAGCCGGTGGAAGAATGACGG-3′), and the reverse primer corresponded to the sequence 4–29 bp downstream from the stop codon of PgFad2 with a flanking KpnI site (5′-atggtaccTTGCGACCAGCAATGTGGTAAATGG-3′). pGEM-T Easy/TkFad2 was digested with SmaI/SacI, and the released cDNA fragment was cloned into the corresponding site of pBluescript II. The TkFad2 cDNA was then released withHindIII/SacI digestion and cloned into pYES2. The PgFad2 cDNA fragment was released from pGEM-T Easy byHindIII/KpnI digestion and cloned into pYES2. The resulting plasmids as well as pYES2 were introduced into S. cerevisiae D452-2 cells (19Matsushita M. Nikawa J. J. Biochem. 1995; 117: 447-451Crossref PubMed Scopus (19) Google Scholar) using an S. c.EasyComp Transformation kit (Invitrogen). Transformed cells were selected for on yeast synthetic minimal medium plates lacking uracil (SC-URA) (20Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Albright L.M. Coen D.M. Varki A. Current Protocols in Molecular Biology. John Wiley & Sons, NY1995Google Scholar). Individual colonies of the transformed cells were grown in glucose culture medium (SC-URA/2% glucose) for 1 day at 28 °C with shaking. Cells were then collected by centrifugation, washed in sterilized water, and dissolved in galactose culture medium (SC-URA/2% galactose). The cell suspension was diluted toA 600 = 0.2 in galactose culture medium containing 0.1% (w/v) Tergitol-type Nonidet P-40 (Sigma) and grown at 20 °C for 3 days followed by 15 °C for 3 days with shaking in the presence or absence of 0.3 mm linoleic, α-linolenic, or 11,14-eicosadienoic acid (20:2Δ11cis, 14cis). Yeast cells were harvested by centrifugation and then washed twice in 1% (w/v) Tergitol solution and three times in distilled water. The washed cells were aliquoted into glass tubes and freeze-dried under vacuum. The dried cell pellets were then incubated with 0.5 m sodium methoxide in methanol at 50 °C for 1 h. After the tubes were cooled to room temperature, the mixture was extracted with hexane. The pooled extracts were dried under vacuum and then dissolved in a small volume of hexane, and 1 μl was used for gas chromatography (GC) or GC-mass spectrometry (MS) analysis. For fatty acid analysis of Arabidopsis materials, seeds (2 mg) were ground with a mortar and pestle and then added to 1 ml of 0.5 m sodium methoxide in methanol. Leaf tissues (4 mg) were homogenized in 1 ml of 0.5 m sodium methoxide in methanol. For T. kirilowii and P. granatum, 2 mg of seeds without seed coats were used for fatty acid extraction. The homogenates were transferred to glass tubes and incubated at 50 °C for 1 h. After the tubes were cooled to room temperature, 1.5 ml of 0.9% (w/v) sodium chloride was added to the samples, and fatty acid methyl esters were extracted with 1 ml of hexane. After centrifugation at 1,000 × g for 5 min, the hexane layer was transferred to a new tube and dried under vacuum. The dried samples were dissolved in a small volume of hexane, and 1 μl was used for GC or GC-MS analysis. Fatty acid methyl esters were analyzed and quantified using a gas chromatograph (GC18A, Shimadzu) equipped with a TC-70 fused silica column (60 m × 0.25-mm inner diameter, 0.25-mm film thickness; GL Science). The oven temperature was programmed to rise from 150 °C to 240 °C at a rate of 3 °C/min and then hold for 6 min. GC-MS analysis was performed in standard EI mode using a JMS-600H MSroute mass spectrometer (JEOL) coupled to a 6890 Series gas chromatograph (Agilent). Samples were separated under conditions as described above. Total fatty acids extracted from maturing seeds of T. kirilowiiand P. granatum were analyzed by GC. Both seeds contained punicic acid at levels of more than ∼40% (w/w) (T. kirilowii) and ∼70% (w/w) (P. granatum) of total fatty acids (see Figs. 2 D and 4 D). We prepared RNA from these materials and isolated four cDNAs from T. kirilowii (TkFad2 and TkFac) and P. granatum (PgFad2 and PgFac) that encode polypeptides related to FAD2. These polypeptides contain three clusters of histidine residues that are thought to act as ligands to the catalytic iron atoms that have been proposed to form a di-iron oxo group (21Shanklin J. Whittle E. Fox B.G. Biochemistry. 1994; 33: 12787-12794Crossref PubMed Scopus (638) Google Scholar). Comparison of amino acid sequences of FAD2-related fatty acid-modifying enzymes (GenBankTMaccession numbers: hydroxylase, T09839 and AAC32755; epoxygenase,CAA76156; acetylenase, CAA76158; and conjugase, AAF05915, AAF05916,AAG42259, AAG42260, and AAK26632) with Δ12-oleate desaturases (GenBankTM accession numbers: P46313, T14269, CAA76157,AAK26633, and AAF78778) revealed that several amino acids at certain positions were strictly conserved in Δ12-oleate desaturases from a number of plant species. As these amino acids were conserved in TkFad2 and PgFad2, we supposed that these were Δ12-oleate desaturases (Fig. 1 A). On the other hand, the phylogenetic analysis indicated that TkFac and PgFac were grouped within a conjugase branch (Fig. 1 B), suggesting that they were conjugases. TkFad2 and PgFad2 encode 369 and 387 amino acids and share 63–68 and 68–71% amino acid identity, respectively, with the Δ12-oleate desaturases. On the other hand, TkFac and PgFac encode 387 and 395 amino acids and share 59–62 and 55–58% amino acid identity to Δ12-oleate desaturases, respectively. PgFac shares 42–54% identity to other conjugases (MomoFadX from M. charantia(7Cahoon 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 (163) Google Scholar), ImpFadX from Impatiens balsamina (7Cahoon 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 (163) Google Scholar), and CoFac2/CoFADXs from C. officinalis (8Cahoon 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 (95) Google Scholar, 9Qiu X. Reed D.W. Hong H. MacKenzie S.L. Covello P.S. Plant Physiol. 2001; 125: 847-855Crossref PubMed Scopus (42) Google Scholar)). TkFac shares sequence identity of 45% to CoFac2/CoFADXs, 55% to ImpFADX, and 55% to PgFac but 74% to MomoFadX. The higher sequence identity between TkFac and MomoFadX may reflect that the two species belong to the same family (Cucurbitaceae).Figure 4Gas chromatographic analyses of FAMEs from yeast cells expressing TkFac and PgFac that were grown in the presence of exogenous linoleic acid. FAMEs from pYES2-transformed (A), TkFac-transformed (B), and PgFac-transformed cells (C) were analyzed by GC. Similarly, FAMEs fromP. granatum seeds were analyzed for reference (D). The labeled peaks corresponding to methyl esters of fatty acids are as follows: 16:0, palmitic acid; 16:1, palmitoleic acid; 16:2, hexadecadienoic acid (Δ9cis, 12cis); 18:0, stearic acid; 18:1, oleic acid; and 18:2, linoleic acid. Arrows, methyl punicic acid; arrowhead, a methyl ester of 16:3 isomer, which was tentatively identified as 16:3Δ9cis, 11trans, 13cis.View Large Image Figure ViewerDownload (PPT)Figure 1Sequence analyses of FAD2-related proteins from T. kirilowii and P. granatum. A, comparison of amino acid sequences of FAD2-related proteins from T. kirilowii(TkFad2 and TkFac) and P. granatum(PgFad2 and PgFac) with FAD2 (Δ12-oleate desaturase) of A. thaliana (AtFad2). Three clusters of histidine residues are indicated by bars. Amino acids identical to those in the TkFac sequence are indicated byshading. Gaps in alignments are indicated bydashes. B, phylogenetic analysis of FAD2 and conjugase proteins using the ClustalW program. The distance along the horizontal axis corresponds to the extent of sequence divergence. Sequences were obtained from GenBankTM accession numbers:ImpFadX, I. balsamina conjugase, AAF05915.1;CoFadX1, CoFadX2, and CoFac2, C. officinalis conjugases, AAG42259, AAG42260, and AAK26632;MomoFadX, M. charantia conjugase, AAF05916.1;CpFad2, Crepis palaestina Δ12-oleate desaturase, CAA76157.1; CoFad2, C. officinalisΔ12-oleate desaturase, AAK26633; and AtFad2, A. thaliana Δ12-oleate desaturase, P46313.View Large Image Figure ViewerDownload (PPT) To examine whether TkFac and PgFac are associated with the formation of punicic acid, the full-length cDNAs encoding TkFac and PgFac were expressed inArabidopsis plants under control of the constitutive CaMV 35S promoter (pKS-TkFac and pKS-PgFac) or the seed-specific napin promoter (pKN-TkFac and pKN-PgFac). Transformants (T1) were selected by drug resistance and by PCR, and then fatty acid methyl esters (FAMEs) from leaves of T1 plants were analyzed by gas chromatography. Although there was no difference in the fatty acid composition of vegetative tissues between transgenic and untransformed plants (data not shown), a prominent peak of FAME (Fig. 2,B and C, arrows), which was not present in seeds from untransformed plant, was found in seeds of transgenic plants. The peak displayed a gas chromatographic retention time identical to that of the methyl ester of punicic acid fromT. kirilowii (Fig. 2 D) and different from those of α-eleostearic and calendic acids extracted from M. charantia and C. officinalis seeds, respectively (data not shown). The mass spectrum of this FAME was identical to that of the methyl punicic acid from T. kirilowii: the molecular ion wasm/z = 292, which was identified as a methyl ester of an isomer of octadecatrienoic acid (18:3), and other diagnostic ions were clearly observed (data not shown). From these results, we concluded that TkFac and PgFac are "conjugases" associated with the formation of the conjugated trans-Δ11,cis-Δ13 double bonds of punicic acid. In addition to the peak of methyl punicic acid, an additional small peak of FAME (Fig. 2,B and C, arrowheads), which was not found in seeds from untransformed plants, was detected in those from transgenic plants. The mass spectrum of this FAME was characterized by an abundant molecular ion at m/z = 320 and other diagnostic ions, which was identified as a methyl ester of an isomer of eicosatrienoic acid (20:3). This peak displayed a gas chromatographic retention time different from that of the methyl 20:3Δ11cis, 14cis, 17cispresent in both untransformed and transformed Arabidopsisseeds (Fig. 2). We analyzed FAMEs of yeast cells fed with 20:2Δ11cis, 14cis to test whether this fatty acid was utilized as a substrate of TkFac and PgFac to produce conjugated 20:3 isomer. However, we could not find a peak corresponding to the methyl 20:3 isomer found in Arabidopsisseeds (data not shown). We supposed that a part of punicic acid was elongated to form the conjugated 20:3 (20:3Δ11cis, 13trans, 15cis) as Arabidopsis seeds possess microsomal fatty acid elongase activity (22James D.W. Lim E. Keller J. Plooy I. Ralston E. Dooner H.K. Plant Cell. 1995; 7: 309-319Crossref PubMed Scopus (312) Google Scholar, 23Millar A.A. Kunst L. Plant J. 1997; 12: 121-131Crossref PubMed Scopus (306) Google Scholar). In all transgenic plants, punicic acid was detected in seeds but not in vegetative tissues (data not shown). We compared the fatty acid composition of seed oils in transgenic and untransformed plants (Table I). Maximal concentration of punicic acid in seed oils was 10.2% (w/w) in pKN-TkFac transformants and 4.4% (w/w) in pKN-PgFac transformants (data not shown). The average concentration of punicic acid was higher in seeds from pKN-TkFac and pKN-PgFac transformants carrying the napin promoter, 3.5 and 2.3% (w/w), respectively. In contrast, concentrations in pKS-TkFac and pKS-PgFac transformants were ∼0.4% (w/w). The concentration of the fatty acid tentatively identified as 20:3Δ11cis, 13trans, 15ciswas in proportion to that of punicic acid (Table I) and accumulated to as much as 1% (w/w) of seed oils in the T2 seeds accumulating 10.2% (w/w) punicic acid (data not shown). Punicic acid accumulation inArabidopsis seeds was accompanied by changes in relative amounts of other fatty acids (Table I). The change was remarkable in seeds of transformants with cDNAs driven by the napin promoter. Relative amounts of linoleic (18:2Δ9cis, 12cis) and linolenic acids (18:3Δ9cis, 12cis, 15cis), which were 30% (w/w) and 19% (w/w) in untransformed seeds, were significantly lower in T2 seeds, at 23–24 and 12–15% (w/w), respectively. In contrast, the concentration of oleic acid was higher in seeds of transgenic plants (23–26% (w/w)) than in those of untransformed plants (15% (w/w)).Table IFatty acid composition of Arabidopsis seeds from untransformed plants and from transgenic plants expressing TkFac and PgFacFatty acidUntransformed (n = 6)TkFacPgFac35S (n = 6)Napin (n = 6)35S (n = 9)Napin (n = 9)16:07.6 ± 0.57.6 ± 0.27.2 ± 0.37.3 ± 0.36.9 ± 0.318:02.6 ± 0.42.9 ± 0.13.2 ± 0.12.7 ± 0.12.9 ± 0.118:1Δ9cis15.2 ± 1.516.2 ± 1.922.8 ± 3.719.1 ± 1.826.4 ± 4.218:2Δ9 cis, 12cis30.3 ± 1.028.4 ± 1.323.3 ± 2.528.9 ± 1.224.2 ± 2.918:3Δ9cis, 12cis, 15cis19.4 ± 1.520.0 ± 1.014.6 ± 3.616.1 ± 1.411.5 ± 2.420:01.8 ± 0.31.9 ± 0.11.7 ± 0.21.7 ± 0.11.5 ± 0.120:1Δ11cis16.8 ± 0.917.8 ± 0.618.7 ± 0.817.0 ± 0.917.3 ± 1.020:2Δ11cis, 14cis1.8 ± 0.21.7 ± 0.21.1 ± 0.31.3 ± 0.20.9 ± 0.220:3Δ11cis, 14cis, 17cis0.4 ± 0.10.5 ± 0.10.3 ± 0.10.3 ± 0.10.2 ± 0.122:1Δ13cis1.7 ± 0.31.7 ± 0.11.5 ± 0.11.4 ± 0.11.2 ± 0.2Punicic acidND0.4 ± 0.43.5 ± 3.30.4 ± 0.32.3 ± 1.120:31-aTentatively identified as 20:3Δ11cis, 13trans, 15cis.ND0.1 ± 0.10.4 ± 0.30.1 ± 0.10.2 ± 0.1Other1-bIncludes 18:1Δ11cis, 20:1Δ13cis, and 22:0.<3.0<2.0<2.0<4.0<5.0Values are represented as weight % of the total fatty acids ofArabidopsis seeds. The means ± S.D. were obtained from independent analyses (number in parentheses) of seeds from individual plants. ND, not detected; 35S, CaMV 35S promoter; Napin, B. napus napin promoter.a Tentatively identified as 20:3Δ11cis, 13trans, 15cis.b Includes 18:1Δ11cis, 20:1Δ13cis, and
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