Oxylipin Profiling Reveals the Preferential Stimulation of the 9-Lipoxygenase Pathway in Elicitor-treated Potato Cells
2001; Elsevier BV; Volume: 276; Issue: 9 Linguagem: Inglês
10.1074/jbc.m008606200
ISSN1083-351X
AutoresCornelia Göbel, Ivo Feußner, Axel Schmidt, Dierk Scheel, José Juan Sánchez‐Serrano, Mats Hámberg, Sabine Rosahl,
Tópico(s)Fungal Plant Pathogen Control
ResumoLipoxygenases are key enzymes in the synthesis of oxylipins and play an important role in the response of plants to wounding and pathogen attack. In cultured potato cells treated with elicitor from Phytophthora infestans, the causal agent of late blight disease, transcripts encoding a linoleate 9-lipoxygenase and a linoleate 13-lipoxygenase accumulate. However, lipoxygenase activity assays and oxylipin profiling revealed only increased 9-lipoxygenase activity and formation of products derived therefrom, such as 9-hydroxy octadecadienoic acid and colneleic acid. Furthermore, the 9-lipoxygenase products 9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoic and 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid were identified as novel, elicitor-inducible oxylipins in potato, suggesting a role of these compounds in the defense response against pathogen attack. Neither 13-lipoxygenase activity nor 13-lipoxygenase products were detected in higher amounts in potato cells after elicitation. Thus, formation of products by the 9-lipoxygenase pathway, including the enzymes hydroperoxide reductase, divinyl ether synthase, and epoxy alcohol synthase, is preferentially stimulated in cultured potato cells in response to treatment with P. infestanselicitor. Moreover, elicitor-induced accumulation of desaturase transcripts and increased phospholipase A2 activity after elicitor treatment suggest that substrates for the lipoxygenase pathway might be provided by de novo synthesis and subsequent release from lipids of the endomembrane system. Lipoxygenases are key enzymes in the synthesis of oxylipins and play an important role in the response of plants to wounding and pathogen attack. In cultured potato cells treated with elicitor from Phytophthora infestans, the causal agent of late blight disease, transcripts encoding a linoleate 9-lipoxygenase and a linoleate 13-lipoxygenase accumulate. However, lipoxygenase activity assays and oxylipin profiling revealed only increased 9-lipoxygenase activity and formation of products derived therefrom, such as 9-hydroxy octadecadienoic acid and colneleic acid. Furthermore, the 9-lipoxygenase products 9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoic and 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid were identified as novel, elicitor-inducible oxylipins in potato, suggesting a role of these compounds in the defense response against pathogen attack. Neither 13-lipoxygenase activity nor 13-lipoxygenase products were detected in higher amounts in potato cells after elicitation. Thus, formation of products by the 9-lipoxygenase pathway, including the enzymes hydroperoxide reductase, divinyl ether synthase, and epoxy alcohol synthase, is preferentially stimulated in cultured potato cells in response to treatment with P. infestanselicitor. Moreover, elicitor-induced accumulation of desaturase transcripts and increased phospholipase A2 activity after elicitor treatment suggest that substrates for the lipoxygenase pathway might be provided by de novo synthesis and subsequent release from lipids of the endomembrane system. polyunsaturated fatty acid 4-coumarate:CoA ligase, EC 6.2.1.12 divinylether synthase epoxy alcohol synthase hydroperoxide lyase lipoxygenase, linoleate:oxygen oxidoreductase, EC 1.13.11.12 hydroxy octadecadienoic acid hydroxy octadecatrienoic acid hydroperoxy octadecadienoic acid hydroperoxy octadecatrienoic acid phenylalanine ammonia-lyase, EC 4.3.1.5 phosphatidylcholine phospholipase A2, phosphatidylcholine 2-acylhydrolase, EC3.1.1.4 pathogenesis-related proteins 1 and 10 hydroxycinnamoyl-CoA:tyramineN-(hydroxycinnamoyl)-transferase, EC 2.3.1.110 kilobase(s) base pair(s) polymerase chain reaction reverse transcriptase high pressure liquid chromatography The formation of hydroperoxy derivatives of polyunsaturated fatty acids (PUFAs)1 represents the first step in the synthesis of oxidized PUFAs, the oxylipins. Their formation in plants may occur either by autoxidation or by the action of enzymes. Enzymatic formation of fatty acid hydroperoxides in plants is catalyzed by nonheme iron-containing lipoxygenases (LOXs, EC1.13.11.12 (1Siedow J.N. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 145-188Crossref Scopus (702) Google Scholar, 2Brash A. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1147) Google Scholar)) and by heme-containing α-dioxygenases (3Hamberg M. Sanz A. Castresana C. J. Biol. Chem. 1999; 274: 24503-24513Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In plants, 9- or 13-LOXs have been identified based on their positional specificity in introducing molecular oxygen into linoleic acid or linolenic acid. Their products, the hydroperoxy PUFAs hydroperoxy octadecadienoic acid (HPOD) and hydroperoxy octadecatrienoic acid (HPOT), are substrates of different enzymes within the LOX pathway (see Fig. 1). A peroxygenase and a reductase catalyze the synthesis of hydroxy octadecadienoic acid (HOD) or hydroxy octadecatrienoic acid (HOT) (4Blée E. Prog. Lipid Res. 1998; 37: 33-72Crossref PubMed Scopus (260) Google Scholar), whereas the activity of divinyl ether synthase (DES) leads to formation of vinyl ether containing PUFAs such as colnele(n)ic and etherole(n)ic acids (5Weber H. Chételat A. Caldelari D. Farmer E.E. Plant Cell. 1999; 11: 485-493PubMed Google Scholar). The synthesis of the signaling compound jasmonic acid originates from 13-HPOT by the activity of an allene oxide synthase (EC 4.2.1.92), whereas a hydroperoxide lyase (HPL) catalyzes the formation of ω-oxo fatty acids and aldehydes (6Feussner I. Kühn H. Bornscheuer U.T. Enzymes in Lipid Modification. Wiley-VCH, Weinheim, Germany2000: 309-336Google Scholar). Trihydroxy octadecenoates are synthesized from linoleic acid-derived epoxy alcohols via epoxy alcohol synthase (EAS) and epoxy alcohol hydrolase (7Hamberg M. Lipids. 1999; 34: 1131-1142Crossref PubMed Scopus (91) Google Scholar). Finally, LOX itself can catalyze the synthesis of keto PUFAs. Functional analyses in transgenic plants have shown the importance of LOXs, the downstream enzymes, and the products of the LOX pathway in the plant's response to wounding and pathogen attack. Thus, transgenicArabidopsis plants with decreased levels of 13-LOX do not show the usual rise in jasmonic acid in response to wounding and are deficient in wound-induced vsp transcript accumulation (8Bell E. Creelman R. Mullet J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8675-8679Crossref PubMed Scopus (455) Google Scholar). Increased susceptibility to insect attack was observed in transgenic potato plants with reduced 13-LOX levels (9Royo J. Léon J. Vancanneyt G. Albar J.P. Rosahl S. Ortego F. Castañera P. Sanchez-Serrano J.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1146-1151Crossref PubMed Scopus (151) Google Scholar) and inArabidopsis plants that were deficient in the LOX substrate linolenic acid (10McConn M. Creelman R.A. Bell E. Mullet J.E. Browse J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5473-5477Crossref PubMed Scopus (658) Google Scholar). In addition to altered responses to wounding and insect attack, defense against the fungal root pathogen Pythium mastophorum was also impaired in plants with decreased levels of linolenic acid (11Vijayan P. Shockey J. Lévesque C.A. Cook R.J. Browse J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7209-7214Crossref PubMed Scopus (460) Google Scholar) as well as in the Arabidopsis jar1 mutant, which is insensitive to jasmonate (12Staswick P.E. Yuen G.Y. Lehman C.C. Plant J. 1998; 15: 747-754Crossref PubMed Scopus (300) Google Scholar). Similarly, the jasmonate-response mutantcoi1 exhibits a higher susceptibility to fungal pathogens such as Alternaria brassicicola and Botrytis cinerea (13Thomma B.P.H.J. Eggermont K. Penninckx I.A.M.A. Mauch-Mani B. Vogelsang R. Cammue B.P.A. Broekaert W.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15107-15111Crossref PubMed Scopus (1173) Google Scholar). In contrast to the rather well studied products of the 13-LOX reaction, such as hexenals, traumatin, and jasmonic acid (14Creelman R.A. Mullet J.E. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 355-381Crossref PubMed Scopus (1504) Google Scholar, 15Wasternack C. Parthier B. Trends Plant Sci. 1997; 2: 302-307Abstract Full Text PDF Scopus (3) Google Scholar, 4Blée E. Prog. Lipid Res. 1998; 37: 33-72Crossref PubMed Scopus (260) Google Scholar), 9-LOX products have only recently become the focus of attention. A possible role in the establishment of resistance of potato against late blight, caused by the oomycete Phytophthora infestans, has been suggested for the 9-LOX-derived divinyl ethers colneleic and colnelenic acids. This was based on the observation that they accumulate in potato leaves after fungal infection and that they exhibit antimicrobial activity (5Weber H. Chételat A. Caldelari D. Farmer E.E. Plant Cell. 1999; 11: 485-493PubMed Google Scholar). Recently, the efficient synthesis of isomeric trihydroxy octadecenoates and octadecadienoates via epoxy alcohols derived from the 9-LOX reaction has been shown to occur in potato leaves (7Hamberg M. Lipids. 1999; 34: 1131-1142Crossref PubMed Scopus (91) Google Scholar). The accumulation of 9-hydroperoxy PUFAs in tobacco after initiation of the hypersensitive response by cryptogein points to a role of 9-LOXs in lipid peroxidation during the hypersensitive response (16Rustérucci C. Montillet J. Agnel J. Battesti C. Alonso B. Knoll A. Bessoule J. Etienne P. Suty L. Blein J. Triantaphylides C. J. Biol. Chem. 1999; 274: 36446-36455Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Apart from these correlative data, the importance of 9-LOXs for resistance was demonstrated in tobacco plants in which the elicitor-induced increase in 9-LOX activity was inhibited by expression of antisense constructs (17Rancé I. Fournier J. Esquerré-Tugayé M.-T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6554-6559Crossref PubMed Scopus (183) Google Scholar). In contrast to the resistant wild type plants, transgenic plants were susceptible to infection withPhytophthora parasitica var. nicotianae. Such a conversion of an incompatible interaction into a compatible one suggests a crucial role for the tobacco 9-LOX in conferring the resistance phenotype. In potato, three distinct classes of LOX cDNAs have been described, which encode a tuber-specific 9-LOX (stlox1), possibly located in the cytoplasm, and two wound-inducible, probably chloroplastic 13-LOXs (stlox2 and stlox3 (18Geerts A. Feltkamp D. Rosahl S. Plant Physiol. 1994; 105: 269-277Crossref PubMed Scopus (82) Google Scholar,19Royo J. Vancanneyt G. Pérez A.G. Sanz C. Störmann K. Rosahl S. Sanchez-Serrano J.J. J. Biol. Chem. 1996; 271: 21012-21019Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar)). Although expression of stlox1 has been shown to be induced in potato leaves in response to infection by P. infestans or after application of the elicitor arachidonic acid (20Fidantsef A.L. Bostock R.M. Physiol. Plant. 1998; 102: 257-271Crossref Scopus (25) Google Scholar), no detailed analysis has been performed addressing the question whether individual LOX transcripts and other products of the LOX pathway apart from the divinyl ethers (5Weber H. Chételat A. Caldelari D. Farmer E.E. Plant Cell. 1999; 11: 485-493PubMed Google Scholar) occur specifically in response to pathogen attack in potato. We therefore used our model system of cultured potato cells to determine the expression pattern of the three LOX isoforms in response to treatment with an elicitor fromP. infestans. Furthermore, we performed oxylipin profiling in elicitor-treated potato cells to obtain insight into enzymatic properties of distinct LOX isoforms during conditions of pathogen attack. In addition, enzymes and metabolites upstream from the LOX pathway (Fig. 1) were studied and are discussed with respect to possible functions in plant pathogen interactions. Culturing of potato cells (cv. Desirée), preparation of crude elicitor from P. infestans, and treatment of suspension-cultured potato cells with elicitor was performed as described previously (21Rohwer F. Fritzemeier K.-H. Scheel D. Hahlbrock K. Planta. 1987; 170: 556-561Crossref PubMed Scopus (51) Google Scholar, 22Schmidt A. Grimm R. Schmidt J. Scheel D. Strack D. Rosahl S. J. Biol. Chem. 1999; 274: 4273-4280Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). RNA from suspension-cultured potato cells was isolated as described (22Schmidt A. Grimm R. Schmidt J. Scheel D. Strack D. Rosahl S. J. Biol. Chem. 1999; 274: 4273-4280Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Hybridizations to radioactively labeled fragments of the different cDNA clones were carried out in 5× SSPE, 5× Denhardt's solution, 0.1%SDS, 50%formamide, 100 μg/ml denatured salmon sperm DNA. Filters were washed three times at 60 °C with 3 × SSC, 0.1%SDS. As probes, the following cDNA fragments were used: a 1.4-kb EcoRI fragment fromstlox1 (18Geerts A. Feltkamp D. Rosahl S. Plant Physiol. 1994; 105: 269-277Crossref PubMed Scopus (82) Google Scholar), a 2.2-kb BamHI fragment fromstlox2 (19Royo J. Vancanneyt G. Pérez A.G. Sanz C. Störmann K. Rosahl S. Sanchez-Serrano J.J. J. Biol. Chem. 1996; 271: 21012-21019Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), a 1.8-kb PstI fragment fromstlox3 (19Royo J. Vancanneyt G. Pérez A.G. Sanz C. Störmann K. Rosahl S. Sanchez-Serrano J.J. J. Biol. Chem. 1996; 271: 21012-21019Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), a 0.9-kb EcoRI fragment fromstchiA (23Büchter R. Stromberg A. Schmelzer E. Kombrink E. Plant Mol. Biol. 1997; 35: 749-761Crossref PubMed Scopus (56) Google Scholar), a 1.2-kb EcoRI fragment fromstpal (24Joos H. Hahlbrock K. Eur. J. Biochem. 1992; 204: 621-629Crossref PubMed Scopus (138) Google Scholar), a 2.0-kb EcoRI fragment fromst4cl (25Becker-André M. Schulze-Lefert P. Hahlbrock K. J. Biol. Chem. 1991; 266: 8551-8559Abstract Full Text PDF PubMed Google Scholar), a 0.95-kb fragment from sttht (22Schmidt A. Grimm R. Schmidt J. Scheel D. Strack D. Rosahl S. J. Biol. Chem. 1999; 274: 4273-4280Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), a 0.3-kb EcoRI fragment from stpr1 (kindly provided by C. Kistner), and a 1.3-kb BamHI fragment fromst25srRNA (kindly provided by J. Petters). For the isolation of a PR10-specific probe, a PCR was carried out using potato genomic DNA and two primers, 5′-GGGTGTCACTAGCTATACACATGAGACC-3′ and 5′-CACTTAAGCGTAGACAGAAGGATTGGCG-3′, covering the coding region of the pr10 gene from Solanum tuberosum (GenBank™ accession number M29041). The 800-bp PCR product was cloned into pCR2.1 (Invitrogen, Groningen, Netherlands) and verified by sequence analysis. RNA from potato cells treated with elicitor for 5 h was used in an RT-PCR reaction using the One-Tube-RT-PCR beads from Amersham Pharmacia Biotech (Freiburg) with the following degenerate primers: 5′-TGG GTI AWH GCH CAY GAR TGB GG-3′ and 5′-CCA RTY CCA YTC IGW BGA RTC RTA RTG-3′ ((26Fritsche K. Hornung E. Peitzsch N. Renz A. Feussner I. FEBS Lett. 1999; 462: 249-253Crossref PubMed Scopus (18) Google Scholar) MWG Biotech, Ebersberg). The PCR product was cloned into the pCR2.1 vector using the TA cloning kit (Invitrogen, Groningen, Netherlands) and sequenced on a LI-COR 4200 (MWG Biotech). The partial cDNA clone had an insert of 579 bp with 97%similarity to a Δ12-desaturase cDNA from Solanum commersonii (GenBank™ accession numberX92847). Total protein was extracted with 100 mm sodium phosphate buffer, pH 7.0, from elicitor- and water-treated cultured potato cells. 40 μg of crude protein extracts was subjected to electrophoresis on 10%denaturing SDS-polyacrylamide gels and transferred onto nitrocellulose filters (Schleicher & Schuell, Dassel, Germany) by electroblotting. Detection of lipoxygenase proteins was performed using polyclonal antiserum raised in rabbits against the N-terminal 539 amino acids of StLOX1 expressed in bacteria (18Geerts A. Feltkamp D. Rosahl S. Plant Physiol. 1994; 105: 269-277Crossref PubMed Scopus (82) Google Scholar). Determination of LOX activity was performed with a Clark oxygen electrode as described (18Geerts A. Feltkamp D. Rosahl S. Plant Physiol. 1994; 105: 269-277Crossref PubMed Scopus (82) Google Scholar). For HPLC analysis of LOX activity, tissue extracts obtained from 0.5 g of material were used. The tissue was extracted with 1 ml of 100 mm potassium phosphate buffer, pH 6.0. After continuous shaking for 30 min at 4 °C, insoluble material was pelleted by centrifugation at 10,000 × g for 10 min. Oxygenation of linoleic acid was carried out by incubating different LOX preparations with the substrate (120 μm final concentration) for 30 min at room temperature. The reaction products were extracted with three volumes of a chloroform/methanol mixture (2:1, v/v) according to a previous study (27Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar). After recovery of the organic phase, solvents were evaporated by vacuo and the lipids were reconstituted in 0.1 ml of the HPLC solvent. Analysis by HPLC of the oxygenated linoleic acid derivatives was carried out as described before (28Feussner I. Kühn H. FEBS Lett. 1995; 367: 12-14Crossref PubMed Scopus (33) Google Scholar, 29Feussner I. Balkenhohl T.J. Porzel A. Kuhn H. Wasternack C. J. Biol. Chem. 1997; 272: 21635-21641Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). For PLA2 activity measurements, 0.3 g of ground cells was mixed with 500 μl of 0.1 m Tris-HCl, pH 7.5, 15 μl 0.1m CaCl2, and 2 μl of [14C]PC (l-α-1-palmitoyl-2-linoleoyl-[linoleoyl-1-14C]-phosphatidylcholine; PerkinElmer Life Sciences, Boston, MA) and incubated for 20 min at room temperature. After addition of 75 μl of acetic acid, two chloroform extractions were performed. After evaporation, the samples were subjected to thin-layer chromatography with chloroform:methanol:H2O (65:25:4) as solvent.14C-Labeled linoleic acid and 14C-labeled PC incubated with PLA2 (Sigma, Munich, Germany) were used as controls. The analysis of LOX-derived products from potato was performed as described (30Weichert H. Stenzel I. Berndt E. Wasternack C. Feussner I. FEBS Lett. 1999; 464: 133-137Crossref PubMed Scopus (81) Google Scholar). The identity of colneleic and colnelenic acid was proven by adding 750 μl of methanol and 250 μl of 20%HCl to 10 μg of colneleic acid and incubating it for 30 min at room temperature. After addition of 200 μl of DNPH, the derivatized aldehyde was analyzed as described (31Kohlmann M. Bachmann A. Weichert H. Kolbe A. Balkenhohl T. Wasternack C. Feussner I. Eur. J. Biochem. 1999; 260: 885-895Crossref PubMed Scopus (63) Google Scholar). For the analysis of jasmonic acid, 0.5 g of tissue was essentially extracted and derivatized as described previously (32Mueller M. Brodschelm W. Anal. Biochem. 1994; 218: 425-435Crossref PubMed Scopus (104) Google Scholar). Gas chromatography/mass spectrometry was performed with a Finnigan GCQ gas chromatography/mass spectrometry system equipped with a capillary Rtx-5 column (5%diphenyl/95%polydimethyl siloxane, 30 m × 0.25 mm; 0.25-μm coating thickness; Restek, Germany). Helium was used as the carrier gas (40 cm × s−1). An electron energy of 70 eV, an ion source temperature of 140 °C, and a temperature of 275 °C for the transfer line were used. The samples were measured in the NCI mode using ammonia as reactant gas, and the splitless injection mode (opened after 1 min) with an injector temperature of 250 °C was used. The temperature gradient was 60–180 °C at 25 °C min−1, 180–270 °C at 5 °C min−1, 270 °C for 1 min, 270–300 °C at 10 °C min−1, and 300 °C for 25 min. For the analysis of trihydroxy oxylipins, frozen cells (1 g) were added to 20 ml of ethanol containing 6.5 nmol of [17,17,18,18,18-2H5]-11(R),12(S),13(S)-trihydroxy-9(Z),15(Z)-octadecadienoic acid and homogenized at 0 °C for 3 min with an Ultraturrax operated at maximum speed. The mixture was extracted with two portions of diethyl ether, and the material obtained was loaded onto an aminopropyl Supelclean SPE tube (0.5 g; Supelco, Bellefonte, PA). Elution was performed with 2-propanol-chlorofom (1:2, v/v), diethyl ether-acetic acid (98:2, v/v), and methanol-acetic acid (98:2, v/v). The last-mentioned eluate was taken to dryness, and the residue was treated with diazomethane and trimethylsilylated. Analysis by gas chromatography/mass spectrometry was carried out using a Hewlett-Packard model 5970B mass selective detector connected to a Hewlett-Packard model 5890 gas chromatograph fitted with a SPB-1701 capillary column (length, 15 m; film thickness, 0.25 mm). The initial column temperature was 120 °C and raised at 10 °C/min until 240 °C. Under these conditions, the retention times of the methyl ester/trimethylsilyl ether derivatives of 9,10,11-trihydroxyoctadecadienoate, 9,10,11-trihydroxyoctadecenoate, and deuterium-labeled standard were 14.3, 14.2, and 14.5 min, respectively. The mass spectrometer was operated in the selected ion-monitoring mode using the ions m/z 278 for the deuterium-labeled standard and m/z 271 for the two 9,10,11-trihydroxy derivatives. Standard curves were constructed by plotting the intensities of m/z271/278 versus the molar ratios of known mixtures of 9,10,11-trihydroxy acids and deuterium-labeled standard and used to calculate the amounts of 9,10,11-trihydroxy acids present in samples analyzed. Potato cells grown in suspension respond to elicitation by a crude preparation of P. infestansculture filtrate with the activation of defense genes, for example, those encoding enzymes of the phenylpropanoid pathway (33Schmidt A. Scheel D. Strack D. Planta. 1998; 205: 51-55Crossref Scopus (68) Google Scholar). To determine the expression pattern of three stlox genes from potato, RNA was isolated from cultured potato cells at different time points after elicitor treatment and subjected to Northern analyses (Fig. 2). Both stlox1 andstlox3 mRNA levels increased transiently in response to elicitor treatment, whereas the level of stlox2 transcripts was below the detection limit both in control and elicitor-treated cultures (data not shown). In different experiments, stlox1transcripts were first detected 1 to 2.5 h after elicitation, reaching maximal levels after 5 to 10 h. In contrast,stlox3 mRNAs started to accumulate earlier, being detectable already 30 min after initiation of treatment and declining after 5 h. The time point of induction of stlox3 gene expression is similar to that of the activation of genes encoding enzymes of the phenylpropanoid pathway, such as PAL and 4-CL as well as THT, as has been shown previously (33Schmidt A. Scheel D. Strack D. Planta. 1998; 205: 51-55Crossref Scopus (68) Google Scholar, 22Schmidt A. Grimm R. Schmidt J. Scheel D. Strack D. Rosahl S. J. Biol. Chem. 1999; 274: 4273-4280Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Hybridization to cDNAs encoding PR1 or PR10 showed that expression of the corresponding genes is induced later, i.e. after 5 h. To determine if the increase in stlox transcript levels correlates with higher protein levels, crude protein extracts were prepared from elicitor-treated as well as water-treated cultured potato cells. Immunochemical analyses using a polyclonal antiserum against StLOX1 showed higher levels of LOX protein 10 and 20 h after elicitor treatment compared with water-treated controls (Fig.3). Extracts prepared from cells treated with elicitor for shorter periods, i.e. for 0, 1, 2.5, and 5 h, did not contain detectable amounts of LOX protein (Fig. 3 and data not shown). A LOX enzyme activity assay performed with a Clark oxygen electrode revealed higher linoleic acid-dependent oxygen consumption in extracts of elicitor-treated cells (data not shown). In a second approach using HPLC analyses, both 13-HOD and 9-HOD were detected, but only 9-HOD was measured to significantly higher amounts in extracts from elicited cells. As shown in Fig. 4, 9-LOX activity started to increase 2.5 h after initiation of treatment and reached five times higher levels after 5 and 10 h. A specific increase in 9-LOX activity upon elicitor treatment is also indicated by a corresponding shift in the ratio of 13- to 9-HOD from 33:67 in extracts of control cells to 7:93 after 5 and 10 h of elicitor treatment (Table I). Chiral phase HPLC revealed that more than 90%of 9-HOD was the Senantiomer, indicating that this compound originated from enzymatic conversion, whereas the racemic nature of the 13-HOD analyzed suggested a nonenzymatic origin (data not shown).Table IRelative levels (%) of 9- and 13-HOD in elicitor (P.i.-cf) or water (H2O)-treated potato cellst9-HOD13-HODP.i.-cfH2OP.i.-cfH2Oh%169 ± 667 ± 032 ± 633 ± 02.581 ± 163 ± 020 ± 138 ± 0593 ± 460 ± 67 ± 440 ± 61093 ± 266 ± 78 ± 234 ± 7 Open table in a new tab The specific induction of a 9-LOX activity in elicitor-treated potato cells was expected to be reflected in elevated levels of metabolites of the oxylipin profile specific for corresponding downstream enzymes. Therefore, this profile was recorded in elicitor-treated and nontreated cells. Although hydroperoxy PUFAs survive the work-up procedure to a significant extent (29Feussner I. Balkenhohl T.J. Porzel A. Kuhn H. Wasternack C. J. Biol. Chem. 1997; 272: 21635-21641Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), HPOD and HPOT levels were below the detection limit. Analysis of the amounts of HOD, the representative of the reductase branch, showed a 5-fold increase in 9-HOD levels in elicitor-treated cells (Fig.5 A). 9-HOD started to accumulate between 10 and 20 h after initiation of treatment. Neither 9-HOT nor the 13-LOX-derived metabolites 13-HOD and 13-HOT could be detected. Among the divinyl ethers derived from the DES reaction, colneleic and colnelenic acid, the derivatives of 9-HPOD and 9-HPOT, respectively, have been reported to accumulate in potato leaves after infection withP. infestans (5Weber H. Chételat A. Caldelari D. Farmer E.E. Plant Cell. 1999; 11: 485-493PubMed Google Scholar). In extracts of cultured potato cells, colneleic acid was detectable as well. In addition to the conventional HPLC analysis determining its characteristic UV spectrum, the identification of colneleic acid was confirmed by acidic hydrolysis of the collected substance and the subsequent detection of (2E)-nonenal as its dinitrophenylhydrazone derivative as one fragment (data not shown). Colneleic acid started to accumulate 5 h after initiation of treatment and reached maximal levels of about 680 pmol/g of fresh weight after 20 h (Fig. 5 B). Colnelenic acid, the divinyl ether derived from linolenic acid, and the corresponding derivatives from 13-HPOD or 13-HPOT, etheroleic and etherolenic acid, could not be detected. In potato leaves, a new branch within the LOX pathway, the EAS pathway, has recently been described, which leads to the synthesis of trihydroxy octadecenoates, the trihydroxy derivatives of linoleic acid (7Hamberg M. Lipids. 1999; 34: 1131-1142Crossref PubMed Scopus (91) Google Scholar). The analysis of elicitor-treated cultured potato cells revealed the elicitor-inducible accumulation of 9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoate, together with a smaller increase in 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoate, the corresponding derivative of linolenic acid (Fig. 5, Cand D). Only small amounts of trihydroxy octadecenoates and trihydroxy octadecadienoic acid were measured in extracts of untreated or water-treated cells. In elicitor-treated cells, accumulation started between 2.5 and 5 h after addition of the elicitor, and levels increased up to 30 h. For the trihydroxy derivative of linoleic acid, at least 10-fold higher levels were measured after 30 h (about 240 pmol/g of fresh weight), whereas the amounts of the trihydroxy derivative of linolenic acid (46 pmol/g of fresh weight) were significantly lower. Changes in the level of other 9-LOX-derived products such as products from the HPL branch were not detected, and, in accordance with the absence of 13-LOX activity in elicited potato cells, no changes in levels of 13-LOX-derived products were found. Neither jasmonic acid nor 13-HOD or 13-HOT were detected at increased levels of elicitor treatment. Similarly, no C6-aldehydes such as (3Z)-hexenal were detectable. Oxylipins derived from autoxidation such as 12- and 16-HOT (31Kohlmann M. Bachmann A. Weichert H. Kolbe A. Balkenhohl T. Wasternack C. Feussner I. Eur. J. Biochem. 1999; 260: 885-895Crossref PubMed Scopus (63) Google Scholar, 16Rustérucci C. Montillet J. Agnel J. Battesti C. Alonso B. Knoll A. Bessoule J. Etienne P. Suty L. Blein J. Triantaphylides C. J. Biol. Chem. 1999; 274: 36446-36455Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) could also not be found in our model system. In summary, maximal formation of the oxylipins colneleic acid, 9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoate, 9-HOD, and 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoate was detected. Because oxylipin profiling showed that the major elicitor-inducible oxylipins in potato cells are 9-LOX-derived metabolites of linoleic acid, studies addressing the origin of LOX substrates were performed. Because StLOX1 appears to be a cytosolic enzyme, a microsomal origin of the substrates was assumed. Therefore, a partial cDNA clone exhibiting high similarity to Δ12-acyl lipid desaturases, which catalyze the formation of linoleic acid from oleic acid within the sn2 position of phosphatidylcholine (PC) within the endoplasmic reticulum (34Heinz E. Moore J., T.S. Biosynthesis of Polyunsaturated Fatty Acids. Lipid Metabolism in Plants. CRC Press, London1993: 33-89Google Scholar),
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