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• W2017885675 abstract "Jasmonic acid (JA) is a lipid-derived signal that regulates a wide variety of developmental and defense-related processes in higher plants. JA is synthesized from linolenic acid via an enzymatic pathway that initiates in the plastid and terminates in peroxisomes. The C18 JA precursor 12-oxo-phytodienoic acid (OPDA) is converted in the peroxisome to 3-oxo-2-(2′-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8:0), which subsequently undergoes three rounds of β-oxidation to yield JA. Although most JA biosynthetic enzymes have been identified, several key steps in the pathway remain to be elucidated. To address this knowledge gap, we employed co-expression analysis to identify genes that are coordinately regulated with known JA biosynthetic components in Arabidopsis. Among the candidate genes uncovered by this approach was a 4-coumarate-CoA ligase-like member of the acyl-activating enzyme (AAE) gene family, which we have named OPC-8:0 CoA Ligase1 (OPCL1). In response to wounding, opcl1 null mutants exhibited reduced levels of JA and hyperaccumulation of OPC-8:0. Recombinant OPCL1 was active against both OPDA and OPC-8:0, as well as medium-to-long straight-chain fatty acids. Subcellular localization studies with green fluorescent protein-tagged OPCL1 showed that the protein is targeted to peroxisomes. These findings establish a physiological role for OPCL1 in the activation of JA biosynthetic precursors in leaf peroxisomes, and further indicate that OPC-8:0 is a physiological substrate for the activation step. The results also demonstrate the utility of co-expression analysis for identification of factors that contribute to jasmonate homeostasis. Jasmonic acid (JA) is a lipid-derived signal that regulates a wide variety of developmental and defense-related processes in higher plants. JA is synthesized from linolenic acid via an enzymatic pathway that initiates in the plastid and terminates in peroxisomes. The C18 JA precursor 12-oxo-phytodienoic acid (OPDA) is converted in the peroxisome to 3-oxo-2-(2′-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8:0), which subsequently undergoes three rounds of β-oxidation to yield JA. Although most JA biosynthetic enzymes have been identified, several key steps in the pathway remain to be elucidated. To address this knowledge gap, we employed co-expression analysis to identify genes that are coordinately regulated with known JA biosynthetic components in Arabidopsis. Among the candidate genes uncovered by this approach was a 4-coumarate-CoA ligase-like member of the acyl-activating enzyme (AAE) gene family, which we have named OPC-8:0 CoA Ligase1 (OPCL1). In response to wounding, opcl1 null mutants exhibited reduced levels of JA and hyperaccumulation of OPC-8:0. Recombinant OPCL1 was active against both OPDA and OPC-8:0, as well as medium-to-long straight-chain fatty acids. Subcellular localization studies with green fluorescent protein-tagged OPCL1 showed that the protein is targeted to peroxisomes. These findings establish a physiological role for OPCL1 in the activation of JA biosynthetic precursors in leaf peroxisomes, and further indicate that OPC-8:0 is a physiological substrate for the activation step. The results also demonstrate the utility of co-expression analysis for identification of factors that contribute to jasmonate homeostasis. Oxylipins comprise a group of potent signaling molecules that are derived from oxidative metabolism of polyunsaturated fatty acids. In both animals and higher plants, oxylipins are synthesized in response to developmental and environmental cues and serve important roles in controlling diverse physiological processes. Members of the prostanoid family of lipid mediators have been studied extensively with respect to their synthesis from arachidonic acid and their function in the regulation of cell differentiation, immune responses, and homeostasis in animal systems (1Smith W.L. Am. J. Physiol. 1992; 263: F181-F191PubMed Google Scholar). The study of plant oxylipins has been focused mainly on the jasmonate family of signaling compounds that includes jasmonic acid (JA) 2The abbreviations used are: JA, jasmonic acid; dhJA, dihydrojasmonic acid; OPDA, 12-oxo-phytodienoic acid; dnOPDA, dinor-OPDA; AOS, allene oxide synthase; OPC-8:0, 3-oxo-2-(2′-[Z]-pentenyl)cyclopentane-1-octanoic acid; AAE, acyl-activating enzyme; WT, wild type; F, forward; R, reverse; GC-MS, gas chromatography-mass spectrometry; YFP, yellow fluorescent protein; GFP, green fluorescent protein; 4CL, 4-coumarate:CoA ligase; LACS, long-chain acyl-CoA synthetase; ACX, acyl-CoA oxidase. and its bioactive precursors and derivatives. Genetic analysis has shown that endogenous jasmonates regulate a wide range of developmental processes including root growth, pollen maturation, anther dehiscence, seed production, and glandular trichome development (2Wallis J.G. Browse J. Prog. Lipid Res. 2002; 41: 254-278Crossref PubMed Scopus (240) Google Scholar, 3Li L. Zhao Y. McCaig B.C. Wingerd B.A. Wang J. Whalon M.E. Pichersky E. Howe G.A. Plant Cell. 2004; 16: 126-143Crossref PubMed Scopus (543) Google Scholar, 4Devoto A. Turner J.G. Physiol. Plantarum. 2005; 123: 161-172Crossref Scopus (145) Google Scholar). In addition, it is well established that jasmonates play a central role in regulating plant responses to biotic and abiotic stress (2Wallis J.G. Browse J. Prog. Lipid Res. 2002; 41: 254-278Crossref PubMed Scopus (240) Google Scholar, 5Gfeller A. Farmer E.E. Science. 2004; 306: 1515-1516Crossref PubMed Scopus (21) Google Scholar, 6Halitschke R. Baldwin I.T. J. Plant Growth Regul. 2005; 23: 238-245Crossref Scopus (106) Google Scholar, 7Howe G.A. J. Plant Growth Regul. 2004; 23: 223-237Crossref Scopus (264) Google Scholar, 8Schilmiller A.L. Howe G.A. Curr. Opin. Plant Biol. 2005; 8: 369-377Crossref PubMed Scopus (423) Google Scholar, 9Wasternack C. Stenzel I. Hause B. Hause G. Kutter C. Maucher H. Neumerkel J. Feussner I. Miersch O. J. Plant Physiol. 2006; 163: 297-306Crossref PubMed Scopus (229) Google Scholar). The biosynthesis of JA from linolenic acid (18:3) is initiated in plastids and terminated in peroxisomes (10Vick B. Zimmerman D. Plant Physiol. 1984; 75: 458-461Crossref PubMed Google Scholar). Lipases that release 18:3 from plastid lipids are thought to play an important role in regulating the pathway in response to environmental and developmental cues (11Farmer E.E. Ryan C.A. Plant Cell. 1992; 4: 129-134Crossref PubMed Google Scholar, 12Ishiguro S. Kawai-Oda A. Ueda J. Nishida I. Okada K. Plant Cell. 2001; 13: 2191-2209Crossref PubMed Scopus (696) Google Scholar, 13Schaller F. Schaller A. Stintzi A. J. Plant Growth Regul. 2005; 23: 179-199Crossref Scopus (185) Google Scholar). The first step in the conversion of 18:3 to JA is catalyzed by 13-lipoxygenase. The resulting 13-hydroperoxy fatty acid is transformed to 12-oxo-phytodienoic acid (OPDA) by the sequential action of allene oxide synthase (AOS) and allene oxide cyclase (AOC). A parallel series of reactions converts hexadecatrienoic acid (16:3) to dinor-OPDA (dnOPDA), which is also a JA precursor (14Weber H. Vick B.A. Farmer E.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10473-10478Crossref PubMed Scopus (274) Google Scholar). That large pools of OPDA/dnOPDA are esterified to chloroplast glycerolipids raises the possibility that JA synthesis is controlled, at least in part, by lipases that release these intermediates from plastid lipids (15Stelmach B.A. Muller A. Hennig P. Gebhardt S. Schubert-Zsilavecz M. Weiler E.W. J. Biol. Chem. 2001; 276: 12832-12838Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 16Buseman C.M. Tamura P. Sparks A.A. Baughman E.J. Maatta S. Zhao J. Roth M.R. Esch S.W. Shah J. Williams T.D. Welti R. Plant Physiol. 2006; 142: 28-39Crossref PubMed Scopus (163) Google Scholar). Very little is known about the mechanism of plastid-to-peroxisome trafficking of JA precursors. Evidence indicates that the ATP-binding cassette transporter PXA1 (also known as CTS1 and PED3) is involved in import of OPDA into peroxisomes (17Theodoulou F.L. Job K. Slocombe S.P. Footitt S. Holdsworth M. Baker A. Larson T.R. Graham I.A. Plant Physiol. 2005; 137: 835-840Crossref PubMed Scopus (202) Google Scholar). The peroxisomal enzyme OPDA reductase (OPR3) converts OPDA to 3-oxo-2-(2′-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8:0) (18Schaller F. Biesgen C. Mussig C. Altmann T. Weiler E.W. Planta. 2000; 210: 979-984Crossref PubMed Scopus (293) Google Scholar, 19Strassner J. Schaller F. Frick U.B. Howe G.A. Weiler E.W. Amrhein N. Macheroux P. Schaller A. Plant J. 2002; 32: 585-601Crossref PubMed Scopus (214) Google Scholar). In the final steps of the pathway, three cycles of β-oxidation remove six carbons from the carboxyl side chain of OPC-8:0 to yield JA. In contrast to the general role of β-oxidation in the degradation of arachidonic acid-derived signals in animals, it is now clear that these catabolic reactions are essential for production of bioactive jasmonates that promote plant developmental and defensive processes (2Wallis J.G. Browse J. Prog. Lipid Res. 2002; 41: 254-278Crossref PubMed Scopus (240) Google Scholar, 8Schilmiller A.L. Howe G.A. Curr. Opin. Plant Biol. 2005; 8: 369-377Crossref PubMed Scopus (423) Google Scholar). Moreover, increasing evidence indicates that JA-modifying enzymes such as JA-amido synthetase (i.e. JAR1) and JA methyltransferase play an important role in altering the biological activity of the hormone following its synthesis in peroxisomes (20Staswick P.E. Tiryaki I. Plant Cell. 2004; 16: 2117-2127Crossref PubMed Scopus (823) Google Scholar, 21Seo H.S. Song J.T. Cheong J.J. Lee Y.H. Lee Y.W. Hwang I. Lee J.S. Choi Y.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4788-4793Crossref PubMed Scopus (558) Google Scholar). In addition to JA and its derivatives, C18 biosynthetic precursors such as OPDA exhibit potent biological activity. Among the physiological processes that are proposed to be regulated by endogenous OPDA are the tendril-coiling response of Bryonia (22Weiler E.W. Albrecht T. Groth B. Xia Z.Q. Luxem M. Liss H. Andert L. Spengler P. Phytochemistry. 1993; 32: 591-600Crossref Scopus (176) Google Scholar), production of various secondary metabolites (23Koch T. Krumm T. Jung V. Engelberth J. Boland W. Plant Physiol. 1999; 121: 153-162Crossref PubMed Scopus (227) Google Scholar, 24Fliegmann J. Schuler G. Boland W. Ebel J. Mithofer A. Biol. Chem. 2003; 384: 437-446Crossref PubMed Scopus (53) Google Scholar), and defense against insects and pathogens (25Stintzi A. Weber H. Reymond P. Browse J. Farmer E.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12837-12842Crossref PubMed Scopus (519) Google Scholar). It has also been shown that exogenous OPDA and JA activate the expression of overlapping but distinct sets of target genes (25Stintzi A. Weber H. Reymond P. Browse J. Farmer E.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12837-12842Crossref PubMed Scopus (519) Google Scholar, 26Taki N. Sasaki-Sekimoto Y. Obayashi T. Kikuta A. Kobayashi K. Ainai T. Yagi K. Sakurai N. Suzuki H. Masuda T. Takamiya K. Shibata D. Kobayashi Y. Ohta H. Plant Physiol. 2005; 139: 1268-1283Crossref PubMed Scopus (385) Google Scholar). These observations have led to the idea that diverse jasmonate-signaled responses are controlled by the relative abundance of multiple bioactive compounds including OPDA/dnOPDA, JA, and various derivatives of JA (14Weber H. Vick B.A. Farmer E.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10473-10478Crossref PubMed Scopus (274) Google Scholar). Our current knowledge of the biochemical and cellular processes that determine this so-called oxylipin signature is still in its infancy. Greater insight into this question will require identification of the complete repertoire of enzymes that promote jasmonate synthesis, as well as an understanding of how these components are regulated. Co-expression analysis has emerged as a powerful tool in the search for plant genes that participate in complex biological processes (27Toufighi K. Brady S.M. Austin R. Ly E. Provart N.J. Plant J. 2005; 43: 153-163Crossref PubMed Scopus (583) Google Scholar, 28Persson S. Wei H.R. Milne J. Page G.P. Somerville C.R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8633-8638Crossref PubMed Scopus (484) Google Scholar, 29Zimmermann P. Hirsch-Hoffmann M. Hennig L. Gruissem W. Plant Physiol. 2004; 136: 2621-2632Crossref PubMed Scopus (2064) Google Scholar, 30Nemhauser J.L. Hong F. Chory J. Cell. 2006; 126: 467-475Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar). The fact that many JA biosynthetic genes are coordinately regulated in response to developmental and stress-related cues (31Reymond P. Weber H. Damond M. Farmer E.E. Plant Cell. 2000; 12: 707-720Crossref PubMed Scopus (1011) Google Scholar, 32Sasaki Y. Asamizu E. Shibata D. Nakamura Y. Kaneko T. Awai K. Amagai M. Kuwata C. Tsugane T. Masuda T. Shimada H. Takamiya K. Ohta H. Tabata S. DNA Res. 2001; 8: 153-161Crossref PubMed Scopus (234) Google Scholar, 33Mandaokar A. Thines B. Shin B. Lange B.M. Choi G. Koo Y.J. Yoo Y.J. Choi Y.D. Choi G. Browse J. Plant J. 2006; 46: 984-1008Crossref PubMed Scopus (264) Google Scholar) indicates that this approach may be useful to identify uncharacterized genes that influence jasmonate homeostasis. Here, we report the use of data-mining tools to identify genes in Arabidopsis that are co-expressed with known JA biosynthetic components. Among the candidate genes singled out by this approach was a member of the large family of ATP-dependent acyl-activating enzymes (AAEs) that metabolize a wide range of carboxylic acid substrates. We provide a combination of genetic, biochemical, and cellular evidence indicating that the enzyme encoded by this gene, which was not previously implicated in JA synthesis, catalyzes the formation of CoA derivatives of jasmonate precursors in leaf peroxisomes. We also provide direct evidence that OPC-8:0 is the physiological substrate for the activation step that immediately precedes β-oxidation. Biological Material—Arabidopsis plants were grown in growth chambers maintained at 22 °C with a photoperiod of 16 h light (100 μmol photons m-2 s-1) and 8 h dark. Columbia-0 (Col-0) was used as wild type (WT) for all experiments. All T-DNA insertion lines used in this study were identified from the Sequence-Indexed Library of Insertion Mutations. Seed stock identifiers for the T-DNA insertion lines were as follows: At4g05160, SALK_050214; At5g63380, SALK_003233; At1g20510, SALK_140659 (corresponds to opcl1-1); and At1g20510, SALK_107614 (corresponds to opcl1-2). Seed stocks were obtained from the Arabidopsis Biological Resource Center. Homozygous knock-out lines were selected by PCR-based screening for both the presence of T-DNA insertion and the absence of an intact endogenous gene. PCR reactions to detect the intact endogenous gene were performed with the following forward (F) and reverse (R) gene-specific primer sets: At4g05160, 5′-AGACACCGTAGAGATTCCTC-3′ (1F) and 5′-ATGTGTTCGGACGTGGAATC-3′ (1R); At5g63380, 5′-GGCTCATTGCTTCTCAAGGC-3′ (2F) and 5′-GGAACGCAACGACCGGAGAT-3′ (2R); At1g20510 (opcl1-1), 5′-TCATCGACGCTTCCACAGGT-3′ (3F) and 5′-GAGTCGAACTCGTAGCTTCC-3′ (3R); and At1g20510 (opcl1-2), 5′-TCCGGTTACTGGTCAGATTCTTGG-3′ (3-2F) and 5′-TGGTCATATTTCAACATGGATGCA-3′ (3-2R). PCR reactions to detect the T-DNA insert in each mutant line were performed with the T-DNA left border primer (LBb1, 5′-GCGTGGACCGCTTGCTGCAACT-3′) and the gene-specific primer indicated in supplemental Fig. S1. PCR analysis confirmed the homozygosity of the T-DNA insert in all mutant lines. RNA Blot Analysis—Rosette leaves from 4-week-old plants were either wounded twice across the mid-vein with a hemostat or sprayed with JA (25 ml of a 50 μm solution per 24 plants). Tissue (1 g fresh weight) was collected at the indicated time points and frozen in liquid nitrogen. RNA extraction and Northern blot analysis were performed according to the procedure described by Li et al. (34Li C. Schilmiller A.L. Liu G. Lee G.I. Jayanty S. Sageman C. Vrebalov J. Giovannoni J.J. Yagi K. Kobayashi Y. Howe G.A. Plant Cell. 2005; 17: 971-986Crossref PubMed Scopus (250) Google Scholar). Gene-specific probes were prepared by PCR amplification of the corresponding cDNA clones for At1g20510 (OPCL1; U09223), At4g05160 (U19441), At5g-63380 (U24744), OPR3 (U13428), and Actin-8 (At1g49240). cDNA clones for OPCL1, At4g05160, At5g63380, and OPR3 were obtained from the Arabidopsis Biological Resource Center. For other probes, PCR amplification was performed with Arabidopsis genomic DNA as template. Forward and reverse PCR primers used to generate these probes were as follows: At4g05160, 5′-AATCCGGCTACGGCAGAGAC-3′ (F) and 5′-TTCAACACCTGGAGCAAGCA-3′ (R); At5g63380, 5′-GAGCTCCGGCTTCGATCAAC-3′ (F) and 5′-GGAACGCAACGACCGGAGAT-3′ (R); At1g20510, 5′-CCAATGGCATACGTCGTAAG-3′ (F2) and 5′-ACTGCCATAATCAATCTGAG-3′ (R2); At1g20480, 5′-AAAGCATGCATGGCGGTTAAGCACGGCGT-3′ (F) and 5′-TCCAGCCACCACAGTGTGCAAC-3′ (R); At1g20490, 5′-TGTTGTGGATCGACTGAAAG-3′ (F) and 5′-TGTACAATGCACAAAAGAAACA-3′ (R); At1g20500, 5′-TAGTTTTTGCTTTGCTTGCG-3′ (F) and 5′-TTCACACTAGTCCAAAAACCAT-3′ (R); At5g38120, 5′-GATCTAATCAAATTTGCCATTT-3′ (F) and 5′-GCTCACAACACATGCTACAC-3′ (R); OPR3, XhoI 5′-GGGCTCGAGATGACGGCGGCACAAGGGAA-3′ (F) and SpeI, 5′-CCCACTAGTTCAGAGGCGGGAAAAAGGAG-3′ (R). Jasmonate Measurements—Rosette leaves were wounded and harvested for jasmonate determinations as described above. Fresh leaf tissue (100-200 mg) was transferred to a Fast-prep tube (Qbiogene, Carlsbad, CA) containing Zirmil beads (Saint-Gobain ZirPro, Mountainside, NJ) and 50 ng of dihydrojasmonic acid (dhJA) as an internal standard. Tubes were capped, frozen in liquid nitrogen, and stored at -80 °C until extraction. Jasmonates were extracted and analyzed according to the vapor-phase extraction procedure (35Schmelz E.A. Engelberth J. Tumlinson J.H. Block A. Alborn H.T. Plant J. 2004; 39: 790-808Crossref PubMed Scopus (211) Google Scholar), with minor modifications as described previously (34Li C. Schilmiller A.L. Liu G. Lee G.I. Jayanty S. Sageman C. Vrebalov J. Giovannoni J.J. Yagi K. Kobayashi Y. Howe G.A. Plant Cell. 2005; 17: 971-986Crossref PubMed Scopus (250) Google Scholar). Endogenous JA levels were quantified using a calibration curve for comparing detector responses of MeJA and dhMeJA (36Mueller M.J. Mene-Saffrane L. Grun C. Karg K. Farmer E.E. Plant J. 2006; 45: 472-489Crossref PubMed Scopus (80) Google Scholar). The combined peak area of the cis and trans isomers of each compound was used for quantification. Because recovery of the dhJA internal standard may not accurately reflect the recovery of ODPA and OPC-8:0 from plant tissue, levels of OPDA and OPC-8:0 were expressed as a relative quantity rather than as an absolute value. Specifically, the relative level of OPDA in extracts from WT and opcl1 plants was calculated by dividing the total GC peak area (cis and trans isomers) of OPDA by the total peak area of the dhJA internal standard. Similarly, the relative level of OPC-8:0 in the extracts was calculated by dividing the total GC peak area (cis and trans isomers) of OPC-8:0 by the peak area of the dhJA. The m/z of [M + H]+ ions and retention times of analyzed methyl esters were as follows: JA (m/z = 225), 7.62 and 8.17 min for trans and cis isomers, respectively; dhJA (m/z = 227), 7.68 and 8.20 min for trans and cis isomers, respectively; OPDA (m/z = 307), 34.69 and 35.22 min for trans and cis isomers, respectively; OPC-8:0 (m/z = 309), 33.45 and 34.17 min for trans and cis isomers, respectively. dhJA was synthesized from JA (Sigma) according to the platinum-catalyzed reduction method (36Mueller M.J. Mene-Saffrane L. Grun C. Karg K. Farmer E.E. Plant J. 2006; 45: 472-489Crossref PubMed Scopus (80) Google Scholar). Authentic standards for OPDA and OPC-8:0 were chemically synthesized as previously described (37Ainai T. Matsuumi M. Kobayashi Y. J. Org. Chem. 2003; 68: 7825-7832Crossref PubMed Scopus (41) Google Scholar). Expression and Purification of Recombinant OPCL1—A full-length OPCL1 cDNA clone was PCR-amplified with the primer set of BamHI (5′-CGCGGATCCATGGCTTCAGTGAATTCTCG-3′ (F); BamHI site underlined) and SalI (5′-AAAGTCGACTCAAAGCTTGGAGTTGGAAG-3′ (R); SalI site underlined). The resulting 1641-bp PCR product was ligated into the BamHI and SalI restriction sites of pQE30 (Qiagen, Valencia, CA). This construct adds 12 amino acids (MRGSHHHHHHGS), including a hexahistidine tag, to the N terminus of OPCL1. The plasmid was transformed into Escherichia coli strain M15 competent cells (Qiagen). Expression and purification of recombinant OPCL1 were performed according to the procedure described by Li et al. (34Li C. Schilmiller A.L. Liu G. Lee G.I. Jayanty S. Sageman C. Vrebalov J. Giovannoni J.J. Yagi K. Kobayashi Y. Howe G.A. Plant Cell. 2005; 17: 971-986Crossref PubMed Scopus (250) Google Scholar). A minor modification was that the lysis buffer contained 300 mm NaCl, complete mini-EDTA-free protease inhibitor mixture tablets (five tablets per 40 ml of buffer; Roche Applied Science) and 1 mg/ml lysozyme. Protein measurements were performed with the BCA assay (Pierce) according to the manufacturer's instructions, with bovine serum albumin as a standard. The purity of recombinant OPCL1 as determined by SDS-polyacrylamide gel electrophoresis was estimated to be >95%. Measurement of Acyl-CoA Synthetase Activity—We measured acyl-CoA synthetase activity of recombinant OPCL1 using the coupled enzyme assay (38Ziegler K. Braun K. Bockler A. Fuchs G. Arch. Microbiol. 1987; 149: 62-69Crossref Scopus (69) Google Scholar), with modifications described by Schneider et al. (39Schneider K. Kienow L. Schmelzer E. Colby T. Bartsch M. Miersch O. Wasternack C. Kombrink E. Stuible H.P. J. Biol. Chem. 2005; 280: 13962-13972Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Fatty acid substrates were converted to their ammonium salts by addition of a few drops of concentrated ammonium hydroxide. The resulting solutions were dried under a stream of N2 gas. These stock solutions were diluted in Triton X-100 and added to the enzyme reaction mixture to yield a final concentration of 0.1% Triton X-100 (v/v). Stock solutions of OPDA and OPC-8:0 (in ethanol) were also diluted in Triton X-100, such that the reaction mixture contained 0.1% Triton X-100 and <0.01% ethanol. Reaction mixtures (700 μl) contained 0.1 m Tris-HCl (pH 7.5), 2 mm dithiothreitol, 5 mm ATP, 10 mm MgCl2, 0.5 mm CoA, 0.1 to 0.4 mm NADH, 100 μm fatty acyl substrate, 1 mm phosphoenolpyruvate, and 10 units each of myokinase, pyruvate kinase, and lactate dehydrogenase. The reaction was initiated by adding ∼10 μg of purified OPCL1. Oxidation of NADH was monitored at 340 nm with a Beckman DU530 spectrophotometer (Fullerton, CA). The assay was validated with a commercial acyl-CoA synthetase (Sigma) from Pseudomonas and 14:0 as a substrate. Heat-killed enzyme was used to determine the rate of non-enzymatic NADH oxidation. Alternatively, total lysate obtained from E. coli cells expressing the empty pQE30 vector was subject to the nickel affinity chromatography procedure used to purify OPCL1. A volume (typically 10 μl) of the resulting eluant was used as a mock-enzyme treatment. Pyruvate kinase (from rabbit muscle), myokinase (from chicken muscle), l-lactate dehydrogenase (from rabbit muscle), and fatty acid substrates were obtained from Sigma. 12-OPDA and OPC-8:0 were chemically synthesized as described previously (37Ainai T. Matsuumi M. Kobayashi Y. J. Org. Chem. 2003; 68: 7825-7832Crossref PubMed Scopus (41) Google Scholar) and were >97% pure. GC-MS analysis showed that the ratio of cis- to trans-OPDA isomers was ∼25:1. The ratio of cis- to trans-OPC-8:0 isomers was ∼1.5:1. Construction of Transgenic Plants Expressing Yellow Fluorescent Protein (YFP)-tagged OPCL1—To determine the subcellular localization of OPCL1, we constructed a transgenic line of Arabidopsis expressing a derivative of the yellow fluorescent protein that is fused to the N terminus of OPCL1. The open reading frame of YFP was PCR amplified from the EYFP-Peroxi plasmid (Clontech, Palo Alto, CA) with the primer set of 5′-CGCGGATCCATGGTGAGCAAGGGCGAG-3′ (BamHI site underlined) and 5′-CTTGTACAGCTCGTCCAT-3′. The OPCL1 open reading frame was PCR amplified from the full-length cDNA with the primer set of 5′-GACGAGCTGTACAAGATGGCTTCAGTGAATTCT-3′ (an overhang encoding overlapping sequence with EYFP is underlined) and 5′-GGATCCTCAAAGCTTGGAGTTGGA-3′ (BamHI site underlined). The fusion between YFP and OPCL1 was created in a second PCR in which equimolar amounts of the two products from the first round of PCR were used as template with the primer set of 5′-CGCGGATCCATGGTGAGCAAGGGCGAG-3′ and 5′-GGATCCTCAAAGCTTGGAGTTGGA-3′ (BamHI site underlined). The resulting product was cloned into the BamHI restriction site of a modified pBI121 vector containing a single BamHI site, which places the fusion gene under the control of the cauliflower mosaic virus 35S promoter. This modified pBI121 vector was constructed as follows. Two pairs of complementary primers (pair 1: 5′-GATCCACTAGTCTCGAG-3′ and 5′-ACGTGCTCGAGACTAGTG-3′; pair 2: 5′-CACGTGGTCGACGGTACCGAGCT-3′ and 5′-CGGTACCGTCGACC-3′) were annealed and ligated to a pBI121 vector that was digested with BamHI and SacI. This manipulation removed the GUS gene and introduced a new set of restriction enzyme sites for cloning. The GFP-OPR3 fusion gene was constructed as follows. The open reading frame of OPR3 was PCR amplified from the cDNA template with the primer set of 5′-GGGCTCGAGATGACGGCGGCACAAGGGAA-3′ (XhoI site underlined) and 5′-CCCACTAGTTCAGAGGCGGGAAAAAGGAG-3′ (SpeI site underlined). The resulting product was cloned into the XhoI and SpeI restriction sites of vector pTA7002. A GFP open reading frame containing 5′ and 3′ XhoI overhangs was amplified from plasmid pEGAD (CD3-389, Arabidopsis Biological Resource Center) with the primer set of 5′-AAACTCGAGATGGTGAGCAAGGGCGAGGA-3′ and 5′-AAGCTTCTCGAGCCCGGGGAATTCGG-3′ (XhoI sites underlined). This product was cloned into the XhoI site of the OPR3-containing pTA7002 vector described above. The YFP-OPCL1 and GFP-OPR3 constructs were transformed into Agrobacterium tumefaciens strain C58C1. The floral dip method (40Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar) was used for Agrobacterium-mediated transformation of Arabidopsis Col-0. T1 generation plants expressing YFP-OPCL1 and T2 generation plants expressing green fluorescent protein (GFP)-OPR3 were used for confocal imaging experiments. Confocal Microscopy—Leaf and root tissue of 15-20-day-old transgenic Arabidopsis plants expressing the fluorescent proteins were hand-sectioned with a razor blade and mounted in distilled water between a slide and coverslip. Confocal fluorescence images of the specimens were taken with a Zeiss LSM5 Pascal laser-scanning confocal microscope (Carl Zeiss, Jena, Germany) equipped with an argon laser. Chloroplast autofluorescence was excited with a 488-nm argon laser and was detected after passage through a long-pass 650-nm emission filter. YFP and GFP fluorescence was excited with a 488-nm laser and was detected after passage through a band pass 505-530-nm emission filter. MitoTracker Orange CM-H2TMRos (Molecular Probes Inc.) was used to stain mitochondria according to the manufacturer's instructions. Fluorescence from MitoTracker-treated root tissue was detected with a 543-nm helium neon laser and a band pass 560-615-nm filter. Identification of an AAE Gene That Is Co-regulated with Other JA Biosynthetic Genes—To facilitate the discovery of new genes involved in jasmonate biosynthesis, we took advantage of the fact that many components of the pathway are coordinately regulated in Arabidopsis (30Nemhauser J.L. Hong F. Chory J. Cell. 2006; 126: 467-475Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar, 32Sasaki Y. Asamizu E. Shibata D. Nakamura Y. Kaneko T. Awai K. Amagai M. Kuwata C. Tsugane T. Masuda T. Shimada H. Takamiya K. Ohta H. Tabata S. DNA Res. 2001; 8: 153-161Crossref PubMed Scopus (234) Google Scholar, 41Reymond P. Bodenhausen N. Van Poecke R.M. Krishnamurthy V. Dicke M. Farmer E.E. Plant Cell. 2004; 16: 3132-3147Crossref PubMed Scopus (423) Google Scholar, 42Devoto A. Ellis C. Magusin A. Chang H.S. Chilcott C. Zhu T. Turner J.G. Plant Mol. Biol. 2005; 58: 497-513Crossref PubMed Scopus (248) Google Scholar). The data-mining tool Expression Angler uses publicly available Affymetrix Arabidopsis GeneChip data to compare the expression pattern of a given query gene to all other elements in the data set (27Toufighi K. Brady S.M. Austin R. Ly E. Provart N.J. Plant J. 2005; 43: 153-163Crossref PubMed Scopus (583) Google Scholar). Query of data sets from the Nottingham Arabidopsis Stock Center's microarray data base (NASCArrays) (43Craigon D.J. James N. Okyere J. Higgins J. Jotham J. May S. Nucleic Acids Res. 2004; 32: D575-D577Crossref PubMed Google Scholar) with OPR3 (At2g06050), a well characterized gene involved in the peroxisomal stage of JA synthesis, identified 67 co-expressed genes (Pearson correlation coefficient (r) > 0.75). The complete list of these genes is given in supplemental Table S1. Among these are genes encoding known or putative JA biosynthetic enzymes including 13-lipoxygenases (At1g72520, At1g17420), AOC (At3g25780), and AOS (At5g42650). One of the highest ranking genes (At1g20510) on the list is a member of the 4-coumarate: CoA ligase (4CL)-like family of AAEs (Fig. 1). A potential role for 4CL-like AAEs in JA biosynthesis is supported by two lines of evidence. First, all eight members of this clade contain a peroxisomal targeting signal type 1 motif (44Shockey J.M. Fulda M.S. Browse J. Plant Physiol. 2003; 132: 1065-1076Crossref PubMed Scopus (136) Google Scholar, 45Reumann S. Ma C. Le" @default.
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• W2017885675 title "Identification of a Peroxisomal Acyl-activating Enzyme Involved in the Biosynthesis of Jasmonic Acid in Arabidopsis" @default.
• W2017885675 cites W1954784702 @default.
• W2017885675 cites W1964716897 @default.
• W2017885675 cites W1965450079 @default.
• W2017885675 cites W1968431236 @default.
• W2017885675 cites W1971171842 @default.
• W2017885675 cites W1974699957 @default.
• W2017885675 cites W1986292144 @default.
• W2017885675 cites W1986991612 @default.
• W2017885675 cites W1992537292 @default.
• W2017885675 cites W1994194264 @default.
• W2017885675 cites W2021010520 @default.
• W2017885675 cites W2022416387 @default.
• W2017885675 cites W2033907783 @default.
• W2017885675 cites W2036499041 @default.
• W2017885675 cites W2043420088 @default.
• W2017885675 cites W2047708521 @default.
• W2017885675 cites W2048832982 @default.
• W2017885675 cites W2049088693 @default.
• W2017885675 cites W2055705389 @default.
• W2017885675 cites W2061712981 @default.
• W2017885675 cites W2068618116 @default.
• W2017885675 cites W2069535324 @default.
• W2017885675 cites W2074403192 @default.
• W2017885675 cites W2074743394 @default.
• W2017885675 cites W2085770357 @default.
• W2017885675 cites W2090861897 @default.
• W2017885675 cites W2094395518 @default.
• W2017885675 cites W2096451658 @default.
• W2017885675 cites W2098115340 @default.
• W2017885675 cites W2099239388 @default.
• W2017885675 cites W2101894478 @default.
• W2017885675 cites W2102370248 @default.
• W2017885675 cites W2102758947 @default.
• W2017885675 cites W2104859889 @default.
• W2017885675 cites W2106542800 @default.
• W2017885675 cites W2110591857 @default.
• W2017885675 cites W2113354546 @default.
• W2017885675 cites W2114973337 @default.
• W2017885675 cites W2115328367 @default.
• W2017885675 cites W2116122104 @default.
• W2017885675 cites W2116767244 @default.
• W2017885675 cites W2118278450 @default.
• W2017885675 cites W2119169356 @default.
• W2017885675 cites W2127924626 @default.
• W2017885675 cites W2128233602 @default.
• W2017885675 cites W2128667600 @default.
• W2017885675 cites W2131295201 @default.
• W2017885675 cites W2132886964 @default.
• W2017885675 cites W2137045089 @default.
• W2017885675 cites W2144462468 @default.
• W2017885675 cites W2144748550 @default.
• W2017885675 cites W2147873096 @default.
• W2017885675 cites W2154395422 @default.
• W2017885675 cites W2158049177 @default.
• W2017885675 cites W2163419872 @default.
• W2017885675 cites W2164811390 @default.
• W2017885675 cites W2171224768 @default.
• W2017885675 cites W4230426154 @default.
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