Functional Characterization of Desaturases Involved in the Formation of the Terminal Double Bond of an Unusual 16:3Δ9, 12, 15 Fatty Acid Isolated from Sorghum bicolor Root Hairs
2006; Elsevier BV; Volume: 282; Issue: 7 Linguagem: Inglês
10.1074/jbc.m606343200
ISSN1083-351X
AutoresZhiqiang Pan, Agnes M. Rimando, Scott R. Baerson, Mark Fishbein, Stephen O. Duke,
Tópico(s)Weed Control and Herbicide Applications
ResumoSorgoleone, produced in root hair cells of sorghum (Sorghum bicolor), is likely responsible for much of the allelopathic properties of sorghum root exudates against broadleaf and grass weeds. Previous studies suggest that the biosynthetic pathway of this compound initiates with the synthesis of an unusual 16:3 fatty acid possessing a terminal double bond. The corresponding fatty acyl-CoA serves as a starter unit for polyketide synthases, resulting in the formation of 5-pentadecatrienyl resorcinol. This resorcinolic intermediate is then methylated by an S-adenosylmethionine-dependent O-methyltransferase and subsequently dihydroxylated, yielding the reduced (hydroquinone) form of sorgoleone. To characterize the corresponding enzymes responsible for the biosynthesis of the 16:3 fatty acyl-CoA precursor, we identified and cloned three putative fatty acid desaturases, designated SbDES1, SbDES2, and SbDES3, from an expressed sequence tag (EST) data base prepared from isolated root hairs. Quantitative real-time RT-PCR analyses revealed that these three genes were preferentially expressed in sorghum root hairs where the 16:2 and 16:3 fatty acids were exclusively localized. Heterologous expression of the cDNAs in Saccharomyces cerevisiae revealed that recombinant SbDES2 converted palmitoleic acid (16:1Δ9) to hexadecadienoic acid (16:2Δ9,12), and that recombinant SbDES3 was capable of converting hexadecadienoic acid into hexadecatrienoic acid (16:3Δ9,12,15). Unlike other desaturases reported to date, the double bond introduced by SbDES3 occurred between carbons 15 and 16 resulting in a terminal double bond aliphatic chain. Collectively, the present results strongly suggest that these fatty acid desaturases represent key enzymes involved in the biosynthesis of the allelochemical sorgoleone. Sorgoleone, produced in root hair cells of sorghum (Sorghum bicolor), is likely responsible for much of the allelopathic properties of sorghum root exudates against broadleaf and grass weeds. Previous studies suggest that the biosynthetic pathway of this compound initiates with the synthesis of an unusual 16:3 fatty acid possessing a terminal double bond. The corresponding fatty acyl-CoA serves as a starter unit for polyketide synthases, resulting in the formation of 5-pentadecatrienyl resorcinol. This resorcinolic intermediate is then methylated by an S-adenosylmethionine-dependent O-methyltransferase and subsequently dihydroxylated, yielding the reduced (hydroquinone) form of sorgoleone. To characterize the corresponding enzymes responsible for the biosynthesis of the 16:3 fatty acyl-CoA precursor, we identified and cloned three putative fatty acid desaturases, designated SbDES1, SbDES2, and SbDES3, from an expressed sequence tag (EST) data base prepared from isolated root hairs. Quantitative real-time RT-PCR analyses revealed that these three genes were preferentially expressed in sorghum root hairs where the 16:2 and 16:3 fatty acids were exclusively localized. Heterologous expression of the cDNAs in Saccharomyces cerevisiae revealed that recombinant SbDES2 converted palmitoleic acid (16:1Δ9) to hexadecadienoic acid (16:2Δ9,12), and that recombinant SbDES3 was capable of converting hexadecadienoic acid into hexadecatrienoic acid (16:3Δ9,12,15). Unlike other desaturases reported to date, the double bond introduced by SbDES3 occurred between carbons 15 and 16 resulting in a terminal double bond aliphatic chain. Collectively, the present results strongly suggest that these fatty acid desaturases represent key enzymes involved in the biosynthesis of the allelochemical sorgoleone. Numerous plant species produce phytotoxic secondary metabolites, some of which may play a direct role in allelopathic interactions (1.Duke S.O. Rimando A.M. Baerson S.R. Scheffler B.E. Ota E. Belz R.G. J. Pest. Sci. 2002; 27: 298-306Crossref Scopus (30) Google Scholar, 2.Inderjit Duke S.O. Planta. 2003; 217: 529-539Crossref PubMed Scopus (491) Google Scholar). These interactions often represent a form of chemical warfare occurring between neighboring plants competing for limited light, water, and nutrient resources (2.Inderjit Duke S.O. Planta. 2003; 217: 529-539Crossref PubMed Scopus (491) Google Scholar, 3.Bais H.P. Park S.W. Weir T.L. Callaway R.M. Vivanco J.M. Trends Plant Sci. 2004; 9: 26-32Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Several Sorghum species have been reported to produce phytotoxins, which are exuded from their root systems into the rhizosphere, which suppress the growth of competing species (4.Einhellig F.A. Agron. J. 1996; 88: 886-893Crossref Scopus (314) Google Scholar). Numerous studies have contributed to the discovery and identification of the chemicals that are responsible for this observed allelopathic inhibition. For example, studies on the biologically active components of both water-soluble and water-insoluble exudates from roots of Sorghum bicolor have demonstrated their role in the growth inhibition of lettuce seedlings (Lactuca sativa), as well as a number of important invasive weed species (5.Netzly D.H. Butler L.G. Crop Sci. 1986; 26: 775-778Crossref Google Scholar). The major constituent of these exudates was identified as 2-hydroxy-5-methoxy-3-[(8′Z,11′Z)-8′,11′,14′-pentadecatriene]-p-benzoquinone, referred to as sorgoleone (Fig. 1) (6.Chang M. Netzly D.H. Butler L.G. Lynn D.G. J. Am. Chem. Soc. 1986; 108: 7858-7860Crossref PubMed Scopus (132) Google Scholar). Early reports on the phytotoxicity of sorgoleone indicated that it is a strong inhibitor of CO2-dependent oxygen evolution in plastids (7.Einhellig F.A. Rasmussen J.A. Hejl A.M. Souza I.F. J. Chem. Ecol. 1993; 19: 369-375Crossref PubMed Scopus (119) Google Scholar). Further studies on its mode of action have documented additional effects on both photosynthetic and mitochondrial electron transport (8.Rasmussen J.A. Hejl A.M. Einhellig F.A. Thomas J.A. J. Chem. Ecol. 1992; 18: 197-207Crossref PubMed Scopus (76) Google Scholar, 9.Gonzalez V.M. Kazimir J. Nimbal C.I. Weston Leslie A. Cheniae G.M. J. Agric. Food Chem. 1997; 45: 1415-1421Crossref Scopus (106) Google Scholar, 10.Rimando A.M. Dayan F.E. Czarnota M.A. Weston L.A. Duke S.O. J. Nat. Prod. 1998; 61: 927-930Crossref PubMed Scopus (104) Google Scholar, 11.Czarnota M.A. Paul R.N. Dayan F.E. Nimbal C.I. Weston L.A. Weed Technol. 2001; 15: 813-825Crossref Google Scholar). The herbicidal and allelopathic properties of sorgoleone make the isolation and characterization of the corresponding genes involved in sorgoleone biosynthesis highly desirable, as manipulation of the pathway in sorghum, or genetic modification of other plant species using these genes could provide important insights into the underlying allelochemical interactions involved (12.Duke S.O. Trends Biotechnol. 2003; 21: 192-195Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Sorgoleone biosynthesis is likely exclusive to root hairs, which appear as cytoplasmically dense cells in sorghum, containing large osmiophilic globules deposited between the plasma lemma and cell wall, presumably associated with sorgoleone rhizosecretion (13.Czarnota M.A. Paul R.N. Weston L.A. Duke S.O. Int. J. Plant Sci. 2003; 164: 861-866Crossref Scopus (82) Google Scholar, 14.Czarnota M.A. Rimando A.M. Weston L.A. J. Chem. Ecol. 2003; 29: 2073-2083Crossref PubMed Scopus (131) Google Scholar). Labeling studies have demonstrated that the biosynthesis of sorgoleone involves the convergence of the fatty acid and polyketide pathways (15.Fate G.D. Lynn D.G. J. Am. Chem. Soc. 1996; 118: 11369-11376Crossref Scopus (54) Google Scholar, 16.Dayan F.E. Kagan I.A. Rimando A.M. J. Biol. Chem. 2003; 278: 28607-28611Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), likely through the action of a novel type III polyketide synthase activity utilizing fatty acyl-CoA starter units, resulting in the addition of a quinone head via iterative condensation of acetate extender units. Subsequent modifications of the alkylresorcinol intermediate are mediated by S-adenosylmethionine-dependent O-methyltransferases and possibly P450 monooxygenases, yielding the reduced form of sorgoleone (a hydroquinone). Upon exudation, the less stable hydroquinone rapidly oxidizes to the highly active benzoquinone form, which can persist in soil for extended periods (11.Czarnota M.A. Paul R.N. Dayan F.E. Nimbal C.I. Weston L.A. Weed Technol. 2001; 15: 813-825Crossref Google Scholar, 17.Netzly D.H. Riopel J.L. Ejeta G. Butler L.G. Weed Sci. 1988; 36: 441-446Crossref Google Scholar, 18.Einhellig F.A. Souza I.F. J. Chem. Ecol. 1992; 18: 1-11Crossref PubMed Scopus (173) Google Scholar). A highly unusual characteristic of the sorgoleone molecule is the presence of a terminal double bond in the aliphatic side chain derived from a desaturated 16:3 fatty acyl-CoA precursor. Although the biochemical pathways involved in biosyntheses of many commonly occurring polyunsaturated fatty acids (PUFAs) in plants have been well characterized (19.Behrouzian B. Buist P.H. Curr. Opin. Chem. Biol. 2002; 6: 577-582Crossref PubMed Scopus (59) Google Scholar, 20.Martin C.E. Oh C.S. Kandasamy P. Chellapa R. Vemula M. Biochem. Soc. Trans. 2002; 30: 1080-1082Crossref PubMed Google Scholar, 21.Wallis J.G. Watts J.L. Browse J. Trends Biochem. Sci. 2002; 27: 467Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar), to our knowledge, enzymes capable of performing desaturation reactions producing a terminal double bond have not previously been characterized in any system. In an attempt to isolate genes encoding fatty acid desaturases involved in the sorgoleone biosynthetic pathway, we have utilized an expressed sequence tag (EST) 3The abbreviations used are: EST, expressed sequence tag; GC, gas chromatograph; MS, mass spectrometry; FAME, fatty acid methyl esters; FAD, fatty acid desaturase; NMR, nuclear magnetic resonance; RACE, rapid amplification of cDNA ends; DMOX, 4,4-dimethyloxazoline; ORF, open reading frame. data base containing ∼5,500 sequences randomly selected from a cDNA library prepared from purified S. bicolor (genotype BT×623) root hairs. 4S. R. Baerson, F. E. Franck E. Dayan, A. R. Agnes M. Rimando, N. P. D. Nanayakkara, C. Liu, J. Schröder, M. Fishbein, Z. Pan, I. A. Kagan, L. H. Pratt, M. Cordonnier-Pratt, and S. O. Duke, submitted manuscript. In this report, we describe the cloning and functional characterization of two fatty acid desaturases, designated SbDES2 and SbDES3, which consecutively convert 16:1 fatty acid to 16:3 fatty acid having a terminal double bond, identified from mining of the sorghum root hair EST data base. 5Fatty 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 zth carbon atom counting from the carboxyl group. No designation for the configuration of double bond is used when the double bond in the fatty acid is in cis configuration. Yeast cells overexpressing the SbDES3 cDNA were able to convert 16:3Δ9,12 fatty acid substrate to its 16:3Δ9,12,15 trienoic form, which is considered to be the substrate used by polyketide synthases in the sorgoleone biosynthetic pathway (15.Fate G.D. Lynn D.G. J. Am. Chem. Soc. 1996; 118: 11369-11376Crossref Scopus (54) Google Scholar, 16.Dayan F.E. Kagan I.A. Rimando A.M. J. Biol. Chem. 2003; 278: 28607-28611Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To our knowledge, this is the first plant fatty acid desaturase described to date exhibiting terminal desaturase activity. Furthermore, the tissue-specific accumulation of SbDES2 and SbDES3 transcripts correlates with the accumulation of 16:2 and 16:3 fatty acids in root hairs (as determined by GC/MS), strongly suggesting the participation of these enzymes in the sorgoleone biosynthetic pathway. Plant Material−Mature leaves, stems, and emerging panicles were harvested from ∼2-month-old, greenhouse-grown Sorghum bicolor (cv. BT×623) plants. Immature leaves and shoot apices were isolated from 8-day-old BT×623 seedlings maintained in a growth chamber at 28 °C, 16 h light/8 h dark, 400 μmol/m2 sec intensity. Total root systems and root hairs were isolated from 8-day-old seedlings grown using a capillary mat system (14.Czarnota M.A. Rimando A.M. Weston L.A. J. Chem. Ecol. 2003; 29: 2073-2083Crossref PubMed Scopus (131) Google Scholar). All tissues were collected, then flash-frozen in liquid nitrogen, and kept at –80 °C prior to extraction. Root hairs were isolated according to the method of Bucher et al. (22.Bucher M. Schroeer B. Willmitzer L. Riesmeier J.W. Plant Mol. Biol. 1997; 35: 497-508Crossref PubMed Scopus (58) Google Scholar). RNA Isolation and Quantitive Real-Time RT-PCR−Total RNAs for real-time PCR experiments shown in Fig. 3 were isolated from 50 mg of flash-frozen, pulverized 10-day-old BT×623 seedling tissues using an RNeasy Plant Mini-Kit (Qiagen, Valencia, CA). Quantitative real-time RT-PCR (QRT-RTPCR) reactions were performed in triplicate using a GenAmp® 5700 Sequence Detection System (Applied Biosystems, Foster City, CA) as previously described (23.Baerson S.R. Sanchez-Moreiras A. Pedrol-Bonjoch N. Schulz M. Kagan I.A. Agarwal A.K. Reigosa M.J. Duke S.O. J. Biol. Chem. 2005; 280: 21867-21881Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Isolation of Sorghum cDNA Clones−To obtain full-length cDNA clones, both 5′- and 3′ rapid amplification of cDNA ends (RACE) was carried out using the BD SMART™ RACE cDNA Amplification Kit (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer's instructions. Primer sets for 5′- and 3′-RACE were 2A03: 5′-CCAAGGAGGTGAAGTGGCAG-3′ and 5′-ATACTACCGGGAGCCACACAAG-3′; 25B05: 5′-TCGCTGACGAAATGGTTGAC-3′ and 5′-CCTCCTTGGCGTGTTCCTC-3′; 56D10: 5′-TGGACGATCACCTCAATCCTG-3′ and 5′-CAACAAGTTCTAGCTGCTTGATGC-3′, respectively. Products of the RACE amplifications were resolved on agarose gels, cloned into the pCR®4-TOPO vector (Invitrogen, Carlsbad, CA), and then confirmed by sequence analysis. Full-length cDNAs were then amplified with primer pairs complementary to the 5′- and 3′-ends of ORFs identified in RACE experiments, using Pfu thermostable DNA polymerase (Stratagene, La Jolla, CA) and first-strand cDNA generated from RNA extracted from sorghum root hairs. Several independent isolates from each amplification were sequenced to ensure the authenticity of the ORFs. Phylogenetic Analysis−Amino acid sequences of putative homology to fatty acid desaturases were retrieved from GenBank™ using BLAST with standard default parameters. A data set was assembled from 54 sequences, in addition to the three sequences characterized here (SbDES 1–3). Sequences of the chlorophyte, Chlorella vulgaris, were included to root the phylogeny. Multiple sequence alignments were constructed with ClustalX ver. 1.81 (24.Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35620) Google Scholar). Three parameter sets were investigated to assess sensitivity to gap costs: default (gap opening, 10.0; gap extension, 0.2), (10.0, 1.0), and (1.0, 1.0). The alignments differed somewhat in length (463, 464, and 482 residues, respectively). Phylogenetic estimates were conducted separately for each alignment. The neighbor-joining method (25.Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar) as implemented in PAUP* ver. 4.0 b 10 6D. Swofford (2001) PAUP* Phylogenetic Analysis Using Parsimony (*, other methods), Version 4, Sinauer Associates, Sunderland, MA. was used with default parameters, except that ties were broken randomly. Trees were midpoint rooted and nodal support was estimated by the bootstrap (27.Felsenstein J. Evolution. 1985; 39: 783-791Crossref PubMed Google Scholar), employing 5000 pseudoreplicate data sets. Trees estimated from the three alignments were extremely similar. The second alignment, with a 10:1 ratio of gap opening to extension penalties, was selected for further analysis. However, all interpretations made here would be identical on trees estimated from the other two alignments (not shown). Plasmid Construction for Heterologous Expression in S. cerevisiae−For heterologous expression in yeast, ORFs were cloned as HindIII/XhoI fragments 3′ to the galactose-inducible GAL1 promoter in pYES2 (Invitrogen), yielding the plasmids pYE25B (for SbDES1 overexpression), pYE56D (for SbDES2 overexpression), and pYE2A (for SbDES3 overexpression). All three constructs were then transformed into yeast strain INVSc1 using the lithium acetate method (28.Burke D. Dawson D. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, NY2000: 103-105Google Scholar). To construct additional vectors for co-expression of two ORFs in yeast, the galactose-inducible promoter::SbDES3 (or SbDES1)::terminator expression cassettes were subcloned into the yeast shuttle vector pRS423 containing a his+ marker (29.Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene (Amst.). 1992; 110: 119-122Crossref PubMed Scopus (1438) Google Scholar). To achieve this, expression cassettes were amplified from pYE2A and pYE25B using the PCR primers Lgal (5′-gggcgcgccACGGATTAGAAGCCGCCGAG-3′) and Rcyc (5′-gggcgcGCCGCAAATTAAAGCCTTCGA-3′), cloned into the pCR®4-TOPO vector (Invitrogen) and confirmed by DNA sequence analyses. The expression cassettes were then excised using BssHII (for SbDES3) or AscI (for SbDES1), gel-purified, and subcloned into BssHII or AscI-digested pRS423, yielding pRS2A (containing SbDES3) and pRS25B (containing SbDES1). These plasmids were then transformed into the yeast strain INVSc1 harboring pYE56D as described above. All yeast transformants were confirmed by colony-PCR using gene specific primers, and by further restriction analyses performed using isolated plasmid preparations. Culture Conditions and Recombinant Protein Expression−Transformed yeast cells were grown in synthetic medium (SC-Ura) containing 2% (w/v) glucose as carbon source and 0.67% (w/v) yeast nitrogen base without amino acids. In the case of co-expression, transformed yeast cells were grown in similar media lacking histidine (SC-UH) for the selection of pRS423. Induction of recombinant protein expression was performed as described by Dyer et al. (30.Dyer 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): briefly, overnight cultures were pelleted by centrifugation, and resuspended in induction medium (2% (w/v) galactose, 0.67% (w/v) yeast nitrogen base without amino acids, and 0.1% (v/v) tergitol Nonidet P-40 (Sigma). The cells then were diluted in 20 ml of same medium to obtain an A600 of 0.4. Exogenous free fatty acids (Sigma), when included, were added to the diluted cells at a final concentration of 0.1% (v/v). Yeast cell lines, including empty vector controls, were induced at 20 °C for 48 h prior to harvest, both in the absence or presence of exogenous fatty acids added to cultures at a final concentration of 0.1% (v/v): palmitic acid, palmitoleic acid, oleic acid, linoleic acid, γ-linolenic acid, and α-linolenic acid (Sigma). For SbDES2 and SbDES3 co-expression experiments, yeast cultures were maintained at 30 °C to obtain optimal yields of the 16:3Δ9,12,15 product (Fig. 6, C and F; Table 1).TABLE 1Accumulation of fatty acid products in transformed yeastGeneProducts accumulatedC16:2C16:3C18:3SbDES24.4 (4.2, 4.5)NDaND, not detected.NDaND, not detected.SbDES3NDaND, not detected.NDaND, not detected.2.1 (2.3, 1.9)SbDES2/34.3 ± 0.31.6 ± 0.2NDaND, not detected.a ND, not detected. Open table in a new tab Fatty Acid Analyses−Cellular fatty acids from yeast and plant tissues were extracted according to the method of Peyou-Ndi et al. (31.Peyou-Ndi M.M. Watts J.L. Browse J. Arch. Biochem. Biophys. 2000; 376: 399-408Crossref PubMed Scopus (87) Google Scholar). Fatty acids were analyzed as methyl ester derivatives on an Agilent 6980GC (Agilent Technologies, Foster City, CA) coupled to a JEOL GCMate II mass spectrometer (JEOL USA, Inc., Peabody, MA) using an Omegawax 250™ (Supelco, St. Louis, MO) capillary column, 30 m × 0.25 mm × 0.25 μm film thickness. The fatty acids were identified by comparison of their retention times and mass spectra with those of authentic standards (Sigma). The inlet (splitless), GC interface, and ion chamber temperatures were 250, 250, and 230 °C, respectively. The volume of sample injected was 1 μl. The GC temperature program for analysis of the hexane extracts, with the exception of the root hair sample, was initial 110 °C held for 5 min, raised to 160 °C at a rate of 10 °C/min, raised to 190 °C at the rate of 1 °C/min, raised to 280 °C at the rate of 2 °C/min and held at this temperature for 2 min. The carrier gas was ultra high purity helium, flow rate 1 ml/min. The root hair hexane extract was analyzed using the following temperature program: initial 80 °C held for 3 min, raised to 170 °C at the rate of 10 °C/ min, raised to 200 °C at the rate of 1 °C/min, raised to 280 °C at the rate of 5 °C/min and held at this temperature for 2 min. For further confirmation of double bond positions, methyl esters were converted into their 4,4-dimethyloxazoline (DMOX) derivatives as previously described (32.Fay L. Richli U. J. Chromatogr. 1991; 541: 89-98Crossref Scopus (222) Google Scholar), except that the derivatization reactions were performed at 170 °C for 15 h. DMOX derivatives were analyzed by GC/MS using the same conditions as described above for the analysis of fatty acid methyl ester derivatives. Isolation and Analysis of 9,12,15(9Z,12Z)-Hexadecatrienoic Acid (16:3 FA) by GC/MS and Nuclear Magnetic Resonance (NMR) Spectroscopy−The 16:3 fatty acid generated by yeast cells overexpressing the SbDES2 and SbDES3 cDNAs was extracted with hexane and isolated by thin layer chromatography using aluminum-backed silica F254 plates (10 × 20 cm2, 0.2 mm silica thickness; EM Science) impregnated with AgNO3. The silica gel plates were pretreated with a solution of 5% AgNO3 in MeOH-H2O (95:5, v/v), air-dried for 60 min, placed in an oven at 80 °C for 16 h, and allowed to cool to room temperature prior to use. Extracts were then applied to plates and developed using CH2Cl2:ethyl acetate (88:12). The 16:3 fatty acid migrated as a band of Rf 0.12, which was scraped from the plate, extracted with CHCl3, and then dried under a stream of nitrogen. An aliquot was analyzed by GC/MS using the same conditions as those described above for the yeast and plant tissue extracts. The 16:3 fatty acid was further characterized by 1H NMR spectroscopy (Bruker DRX 400 UltraShield™ spectrometer, XWIN-NMR Software Version 3.1; Bruker, Billerica, MA). 1H NMR (CDCl3): δ 5.80 (m, 1H, H-15); 5.30 (m, 4H, H-9, 10, 12, 13); 4.98 (br d, J = 17 Hz, 1H, H-16a); 5.02 (br d, J = 10 Hz, 1H, H-16b); 4.90–5.01 (br dd, 2H, H-16); 3.71 (s, 3H, OCH3), 2.78 (m, 4H, H-11, 14), 2.32–2.36 (m, 2H, H-2), 2.00 (m, 2H, H-8), 1.49 (m, 2H, H-3), 1.25–1.29 (m, 8H, H-4, 5, 6, 7). Fatty Acid Composition Analyses in Sorghum Tissues−The biosynthesis of the allelochemical sorgoleone is a multistep process involving both plastidic and cytoplasmic enzymes (15.Fate G.D. Lynn D.G. J. Am. Chem. Soc. 1996; 118: 11369-11376Crossref Scopus (54) Google Scholar, 16.Dayan F.E. Kagan I.A. Rimando A.M. J. Biol. Chem. 2003; 278: 28607-28611Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). One of the key steps is the formation of hexadecatrienoic acid (16:3Δ9,12,15), an unsaturated fatty acid proposed to serve as a precursor for the associated PKS in the pathway. To examine the presence and distribution of 16:2Δ9,12 and 16:3Δ9,12,15 in S. bicolor, the fatty acid compositions of various tissues (mature and immature leaves, stems, shoot apices, panicles, and whole root systems) were analyzed (Fig. 2A). The predominance of palmitic (16:0), linoleic (18:2), and linolenic (18:3) acids was observed in all sorghum tissues. Traces of 16:2 (peak at 27.0 min) and 16:3 (peak at 29.7 min) were observed in the chromatograms of extracted roots, as indicated by mass spectral data (not shown); however the low levels for these peaks precluded their unequivocal identification. As sorgoleone is a major component in the oily exudate droplets associated with sorghum root hairs (13.Czarnota M.A. Paul R.N. Weston L.A. Duke S.O. Int. J. Plant Sci. 2003; 164: 861-866Crossref Scopus (82) Google Scholar, 14.Czarnota M.A. Rimando A.M. Weston L.A. J. Chem. Ecol. 2003; 29: 2073-2083Crossref PubMed Scopus (131) Google Scholar), root hairs were also isolated and analyzed for the presence of the precursor C16 fatty acids. Two peaks (corresponding to 16:2 and 16:3 fatty acid) were detected in the lipid extracts (Fig. 2B) by GC/MS. The mass spectrum (Fig. 2C) of the fatty acid methyl ester (FAME) corresponding to the 16:2 peak (Fig. 2B) exhibited a prominent molecular ion at m/z = 266.2, characteristic of a 16:2 methyl ester and identical to that of methyl 9,12-hexadecadienoate, whereas the spectrum of the second peak corresponding to 16:3 (Fig. 2B) had a molecular ion at m/z = 264.1, and a fragmentation pattern consistent with hexadecatrienoate possessing a terminal methylene (Fig. 2D). Collectively, these data identify the 16:3Δ9,12 and 16:3Δ9,12,15 fatty acids in root hairs, and furthermore, strongly suggest that these precursors are primarily synthesized in root hair cells. Identification and Cloning of Fatty Acid Desaturases from Sorghum−To identify the desaturases associated with the sorgoleone biosythetic pathway, particularly those involved in the terminal bond desaturation of C16 fatty acid, an EST data base generated using a cDNA library prepared from isolated sorghum root hair cells was mined. Data base mining was performed both by using the Magic Gene Discovery software (33.Cordonnier-Pratt M.-M. Liang C. Wang H. Kolychev D.S. Sun F. Freeman R. Sullivan R. Pratt L.H. Comp. Funct. Genom. 2004; 5: 268-275Crossref PubMed Scopus (18) Google Scholar) and by BLAST analysis. From these analyses, we identified 47 desaturase-like ESTs, which assembled into 11 unique contigs. Quantitative real-time RT-PCR analysis showed that 3 of 11 unique desaturase sequences (suggested by clustering) were preferentially expressed in root hair cells (Fig. 3). The corresponding full-length cDNA clones were isolated using cDNA prepared from root hair cells, and designated SbDES1, SbDES2, and SbDES3. The protein sequences deduced from SbDES3 and SbDES1 were 90% identical to each other, and shared only 33.7% and 33.8% identity, respectively, with the sequence from SbDES2. BLAST analysis of these protein sequences revealed that both SbDES3 and SbDES1 exhibited significant similarity to known plant fatty acid desaturase (FAD3-type) sequences, and SbDES2 displayed a high degree of similarity to plant FAD2-type sequences (34.Los D.A. Murata N. Biochim. Biophys. Acta. 1998; 1394: 3-15Crossref PubMed Scopus (441) Google Scholar, 35.Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (731) Google Scholar). A previously isolated putative desaturase clone from the S. bicolor × S. sudanense hybrid, SX17 (36.Yang X. Scheffler B.E. Weston L.A. J. Exp. Bot. 2004; 55: 2251-2259Crossref PubMed Scopus (42) Google Scholar), showed homology to the SbDES3 clone described in this article. Based on comparisons to known FADs, the predicted protein sequences of all three cDNAs exhibited all of the major structural features possessed by FADs from other systems (34.Los D.A. Murata N. Biochim. Biophys. Acta. 1998; 1394: 3-15Crossref PubMed Scopus (441) Google Scholar, 35.Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (731) Google Scholar), notably the predicted transmembrane domains and three highly conserved histidine-rich motifs occurring in most membrane-bound desaturases (Fig. 4). The conserved histidine-rich motifs are believed to be required for fatty acid desaturase activity (37.Shanklin J. Whittle E. Fox B.G. Biochemistry. 1994; 33: 12787-12794Crossref PubMed Scopus (650) Google Scholar). These comparisons were further supported by phylogenetic analyses (Fig. 5). SbDES2 was most closely related to members of the plant microsomal FAD2-type desaturase subfamily which, among those functionally characterized, typically desaturate C16/C18 acyl chains at the Δ12 position. In contrast, both SbDES1 and SbDES3 were positioned within the group corresponding to the plant microsomal FAD3-type desaturases, which mainly catalyze the conversion of 18:2 to 18:3 in phospholipids (38.Browse J. McConn M. James Jr., D. Miquel M. J. Biol. Chem. 1993; 268: 16345-16351Abstract Full Text PDF PubMed Google Scholar). For example, the closely related FAD3-like enzymes from wheat and rice (GenBank™ accession nos. BAA28358 and BAA11397- Fig. 5) catalyze the conversion of linoleic acid (18:2Δ9,12) to linolenic acid (18:3Δ9,12,15) (39.Kodama H. Akagi H. Kusumi K. Fujimura T. Iba K. Plant Mol. Biol. 1997; 33: 493-502Crossref PubMed Scopus (52) Google Scholar, 40.Horiguchi G. Fuse T. Kawakami N. Kodama H. Iba K. Plant J. 2000; 24: 805-813Crossref PubMed Google Scholar). Functional Characterization of FAD-like cDNAs in S. cerevisiae−For functional characterization of the putative FADs, vectors were engineered for heterologous expression in S. cerevisiae. The complete ORFs for SbDES1, SbDES2, and SbDES3, were cloned into the yeast expression vector pYES2, resulting in the vectors pYE25B, pYE56D, and pYE2A, respectively. Constructs were transformed into the S. cerevisiae strain INVSc1, and the resulting transformants were cultivated in the presence and absence of exogenously supplied pa
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