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W2108372224 abstract "Lipid peroxidation was investigated in relation with the hypersensitive reaction in cryptogein-elicited tobacco leaves. A massive production of free polyunsaturated fatty acid (PUFA) hydroperoxides dependent on a 9-lipoxygenase (LOX) activity was characterized during the development of leaf necrosis. The process occurred after a lag phase of 12 h, was accompanied by the concomitant increase of 9-LOX activity, and preceded by a transient accumulation of LOX transcripts. Free radical-mediated lipid peroxidation represented 10% of the process. Inhibition and activation of the LOX pathway was shown to inhibit or to activate cell death, and evidence was provided that fatty acid hydroperoxides are able to mimic leaf necrotic symptoms. Within 24 h, about 50% of leaf PUFAs were consumed, chloroplast lipids being the major source of PUFAs. The results minimize the direct participation of active oxygen species from the oxidative burst in membrane lipid peroxidation. They suggest, furthermore, the involvement of lipase activity to provide the free PUFA substrates for LOX. The LOX-dependent peroxidative pathway, responsible for tissue necrosis, appears as being one of the features of hypersensitive programmed cell death. Lipid peroxidation was investigated in relation with the hypersensitive reaction in cryptogein-elicited tobacco leaves. A massive production of free polyunsaturated fatty acid (PUFA) hydroperoxides dependent on a 9-lipoxygenase (LOX) activity was characterized during the development of leaf necrosis. The process occurred after a lag phase of 12 h, was accompanied by the concomitant increase of 9-LOX activity, and preceded by a transient accumulation of LOX transcripts. Free radical-mediated lipid peroxidation represented 10% of the process. Inhibition and activation of the LOX pathway was shown to inhibit or to activate cell death, and evidence was provided that fatty acid hydroperoxides are able to mimic leaf necrotic symptoms. Within 24 h, about 50% of leaf PUFAs were consumed, chloroplast lipids being the major source of PUFAs. The results minimize the direct participation of active oxygen species from the oxidative burst in membrane lipid peroxidation. They suggest, furthermore, the involvement of lipase activity to provide the free PUFA substrates for LOX. The LOX-dependent peroxidative pathway, responsible for tissue necrosis, appears as being one of the features of hypersensitive programmed cell death. hypersensitive reaction programmed cell death active oxygen species lipoxygenase polyunsaturated fatty acid high pressure liquid chromatography jasmonate methyl jasmonate 15-hydroxy-11,13(Z,E)-eicosadienoic acid fresh weight dry weight rapid amplification of cDNA ends polymerase chain reaction palmitic acid palmitoleic acid trans-palmitoleic acid hexadecadienoic acid hexadecatrienoic acid stearic acid oleic acid linoleic acid linolenic acid 9-hydroxy-10,12(Z,E)-octadecadienoic acid 9-hydroxy-10,12 (E,E) octadecadienoic acid 13-hydroxy-9,11(Z,E)-octadecadienoic acid 13-hydroxy-9,11(E,E) octadecadienoic acid 9-hydroxy-10,12,15(E,Z,Z) octadecatrienoic acid 12-hydroxy-9,13,15,(Z,E,Z)-octadecatrienoic acid 13-hydroxy-9,11,15(Z,E,Z)-octadecatrienoic acid 16-hydroxy-9,12,14,(Z,Z,E)-octadecatrienoic acid 9-hydroperoxy-10,12(Z,E)-octadecadienoic acid 13-hydroperoxy-9,11(Z,E)-octadecadienoic acid 9-hydroperoxy-10,12,15(E,Z,Z) octadecatrienoic acid 13-hydroperoxy-9,11,15(Z,E,Z)-octadecatrienoic acid tert-butyl hydroperoxide In plant-pathogen interactions, a typical feature of plant resistance is hypersensitive reaction (HR),1 characterized by the induction of rapid cell death at the site of an attempted attack by either an avirulent strain of a pathogen or a non-pathogen. The collapse of challenged cells, occurring during incompatible interactions, was shown in most cases to be dependent on a gene for gene plant pathogen interaction (1Greenberg J.T. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 525-545Crossref PubMed Scopus (423) Google Scholar, 2Pontier D. Balagué C. Roby D. C. R. Acad. Sci. (Paris). 1998; 321: 721-734Crossref PubMed Scopus (85) Google Scholar). HR is accompanied by a battery of defense mechanisms including de novo synthesis of antimicrobial enzymes and metabolites, strengthening of the cell wall, and the onset of systemic acquired resistance dependent on salicylic acid accumulation (3Bowles D.J. Annu. Rev. Biochem. 1990; 59: 873-907Crossref PubMed Scopus (872) Google Scholar, 4Hunt M.D. Ryals J.A. Crit. Rev. Plant Sci. 1996; 15: 583-606Crossref Google Scholar). HR often leads to dry lesions that are supposed to limit pathogen growth. Other proposed roles is the release in apoplasm of defense-related proteins and toxic metabolites, as well as of signals that activate the defenses of both neighboring and distant cells. Hypersensitive cell death appears to not be the result of the direct action of released pathogenic factors but is rather under the genetic control of the host. Indeed, several observations underline that HR is an example of PCD in plants (1Greenberg J.T. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 525-545Crossref PubMed Scopus (423) Google Scholar, 2Pontier D. Balagué C. Roby D. C. R. Acad. Sci. (Paris). 1998; 321: 721-734Crossref PubMed Scopus (85) Google Scholar). Furthermore, hypersensitive cell death has morphological and molecular features similar to the mammalian PCD, called apoptosis. These include cytoplasm and chromatin condensation followed by their fragmentation, activation of calcium-dependent endonucleases (5Mittler R. Lam E. Plant Cell. 1995; 7: 1951-1962Crossref PubMed Scopus (172) Google Scholar, 6Levine A. Pennell R.I. Alvarez M.E. Palmer R. Lamb C. Curr. Biol. 1996; 6: 427-437Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 7Ryerson D.E. Heath M.C. Plant Cell. 1996; 8: 393-402Crossref PubMed Google Scholar, 8Mittler R. Simon L. Lam E. J. Cell Sci. 1997; 110: 1333-1344PubMed Google Scholar) and of cysteine proteases (9del Pozo O. Lam E. Curr. Biol. 1998; 8: 1129-1132Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 10D'Silva I. Poirier G.G. Heath M.C. Exp. Cell Res. 1998; 245: 389-399Crossref PubMed Scopus (87) Google Scholar, 11Solomon M. Benleghi B. Delledonne M. Levine A. Plant Cell. 1999; 11: 431-444Crossref PubMed Scopus (654) Google Scholar), and involvement of similar regulation factors (2Pontier D. Balagué C. Roby D. C. R. Acad. Sci. (Paris). 1998; 321: 721-734Crossref PubMed Scopus (85) Google Scholar). Some differences between HR and mammalian apoptosis were observed, however, such as changes in DNA laddering (5Mittler R. Lam E. Plant Cell. 1995; 7: 1951-1962Crossref PubMed Scopus (172) Google Scholar, 8Mittler R. Simon L. Lam E. J. Cell Sci. 1997; 110: 1333-1344PubMed Google Scholar) and the lack in HR of the repressor role of Bcl-x L (12Mittler R. Shulaev V. Seskar M. Lam E. Plant Cell. 1996; 8: 1991-2001Crossref PubMed Scopus (114) Google Scholar). One ultimate characteristic of HR is the loss of membrane integrity, and thus HR is often characterized by an associated electrolyte leakage (5Mittler R. Lam E. Plant Cell. 1995; 7: 1951-1962Crossref PubMed Scopus (172) Google Scholar, 13Pike S.M. Adam A.L. Pu X.A. Hoyos M.E. Laby R. Beer S.V. Novacky A. Physiol. Mol. Plant Pathol. 1998; 53: 39-60Crossref Scopus (47) Google Scholar). This feature is not encountered in mammalian apoptosis but is one characteristic of the catastrophic cell death called necrosis, which is not dependent on gene activation (14Malcomson R.D.G. Oram S.H. Harrison D.J. Biologicals. 1996; 24: 295-299Crossref PubMed Scopus (4) Google Scholar). In this way, the use of the term “necrosis” assumes different meanings when referring to mammalian cell death or to pathogen-associated plant cell death. Membrane damage during HR is in close correlation with lipid peroxide production and with AOS generation (12Mittler R. Shulaev V. Seskar M. Lam E. Plant Cell. 1996; 8: 1991-2001Crossref PubMed Scopus (114) Google Scholar, 15Adam A. Farkas T. Somlyai G. Hevesi M. Kiraly Z. Physiol. Mol. Plant Pathol. 1989; 34: 13-26Crossref Scopus (196) Google Scholar, 16May M.J. Hammond-Kosack K.E. Jones J.D.G. Plant Physiol. (Rockv.). 1996; 110: 1367-1379Crossref PubMed Scopus (180) Google Scholar, 17Rustérucci C. Blein J.-P. Stallaert V. Ducruet J.-M. Pugin A. Ricci P. Plant Physiol. (Rockv.). 1996; 111: 885-891Crossref PubMed Scopus (130) Google Scholar). AOS can initiate lipid peroxidation in membranes by fatty acid free radical production, and the process can be propagated by autoxidation (18Porter N.A. Caldwell S.A. Mills K.A. Lipids. 1995; 30: 277-289Crossref PubMed Scopus (1020) Google Scholar). The generation of AOS during the oxidative burst is an important early event during the course of plant-pathogen interactions and is well documented (19Doke N. Scandalios J.G. Oxidative Stress and the Molecular Biology of Antioxidant Defenses. 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 785-813Google Scholar, 20Wojtaszek P. Biochem. J. 1997; 322: 681-692Crossref PubMed Scopus (1035) Google Scholar). In the HR, lipid peroxidation is often a late process occurring at the same time as the appearance of necrosis. Since AOS production preceded lipid peroxidation, it is generally admitted that AOS are implicated in the initiation of membrane damage, and hence hypersensitive cell death. Indeed, inhibition of oxidative burst by exogenous supplied enzymes, scavengers, or inhibitors of AOS generator systems suppresses or delays both lipid peroxidation and hypersensitive cell death (15Adam A. Farkas T. Somlyai G. Hevesi M. Kiraly Z. Physiol. Mol. Plant Pathol. 1989; 34: 13-26Crossref Scopus (196) Google Scholar, 21Doke N. Ohashi Y. Physiol. Mol. Plant Pathol. 1988; 32: 163-175Crossref Scopus (200) Google Scholar, 22Tenhaken R. Levine A. Brisson L.F. Dixon R.A. Lamb C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4158-4163Crossref PubMed Scopus (350) Google Scholar, 23Jabs T. Dietrich R.A. Dangl J.L. Science. 1996; 273: 1853-1856Crossref PubMed Scopus (700) Google Scholar). Lipid peroxidation might also be due to LOX (EC 1.13.11.12) activity (24Hildebrand D.F. Physiol. Plant. (Rockv.). 1989; 76: 249-253Crossref Scopus (252) Google Scholar, 25Rosahl S. Z. Naturforsh. 1996; 51: 123-138Crossref PubMed Scopus (124) Google Scholar). Initiation of HR membrane damage by LOXs has been suggested as an alternative hypothesis to free radical action, and the process might be propagated by autoxidation (26Croft K.P.C. Voisey C.R. Slusarenko A.J. Physiol. Mol. Plant Pathol. 1990; 36: 49-62Crossref Scopus (138) Google Scholar, 27Kondo Y. Kawai Y. Hayashi T. Ohnishi M. Miyazawa T. Itoh S. Mizutani J. Biochim. Biophys. Acta. 1993; 1170: 301-306Crossref PubMed Scopus (31) Google Scholar). Indeed, induction of LOX activity has been observed in several plants during incompatible interaction and occurs after a lag phase of few hours (28Slusarenko A.J. Piazza G. Lipoxygenase and Lipoxygenase Pathway Enzymes. AOCS Press, Champaign, IL1996: 176-197Crossref Google Scholar). The observation that an incompatible interaction can be suppressed in transgenic tobacco plants expressing antisense LOX clearly demonstrates the role of LOX in plant resistance to pathogens (29Rancé I. Fournier J. Esquerré-Tugayé M.-T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6554-6559Crossref PubMed Scopus (185) Google Scholar). Finally, since (i) the LOX pathway leads to products, such as hydroperoxides, alkenals, and aldehydes, that may kill plant cells and pathogen (30Farmer E.E. Plant Mol. Biol. 1994; 26: 1423-1437Crossref PubMed Scopus (179) Google Scholar,31Farmer E.E. Weber H. Vollenweider S. Planta. 1998; 206: 167-174Crossref PubMed Scopus (133) Google Scholar) and (ii) HR triggering is an example of PCD, the induction of a LOX pathway could be considered as an active process of membrane degradation leading to plant cell death. Thus, it is not clear at present whether lipid peroxidation during HR is induced by AOS and free radicals or is the result of a LOX action. Both mechanisms could operate in parallel or be exclusive. Furthermore, the question of whether membrane lipid peroxidation induces cell death or is the consequence of cell death is still open. The HR induced in tobacco (Nicotiana tabacum) leaves by cryptogein, a purified protein from the fungus Phytophthora cryptogea (32Ricci P. Bonnet P. Huet J.-C. Sallantin M. Beauvais-Cante F. Bruneteau M. Billard V. Michel G. Pernollet J.-C. Eur. J. Biochem. 1989; 183: 555-563Crossref PubMed Scopus (280) Google Scholar), was investigated in this work. Cryptogein leads also to defense gene activation (33Suty L. Petitot A.-S. Lecourieux D. Blein J.-P. Pugin A. Plant Physiol. Biochem. 1996; 34: 443-451Google Scholar) and systemic acquired resistance (34Bonnet P. Bourdon E. Ponchet M. Blein J.-P. Ricci P. Eur. J. Plant Pathol. 1996; 102: 181-192Crossref Scopus (132) Google Scholar). Features of PCD were observed, as assessed by plasma membrane blebbing, cell shrinkage, and cytoplasmic condensation (6Levine A. Pennell R.I. Alvarez M.E. Palmer R. Lamb C. Curr. Biol. 1996; 6: 427-437Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar), and the expression of hsr 203J, a gene proposed as the hallmark of HR-inducing pathogens or elicitors (35Pontier D. Tronchet M. Rogowsky P. Lam E. Roby D. Mol. Plant-Microbe Interact. 1998; 11: 544-554Crossref PubMed Scopus (139) Google Scholar). On tobacco cells cryptogein induces an early oxidative burst (36Simon-Plas F. Rustérucci C. Milat M.-L. Humbert C. Montillet J.-L. Blein J.-P. Plant Cell Environ. 1997; 20: 1573-1579Crossref Scopus (62) Google Scholar) and late LOX activity (37Bottin A. Véronési C. Pontier D. Esquerré-Tugayé M.-T. Blein J.-P. Rustérucci C. Ricci P. Plant Physiol. Biochem. 1994; 32: 373-378Google Scholar). An early AOS production was also characterized on leaves (38Allan A.C. Fluhr R. Plant Cell. 1997; 9: 1559-1572Crossref PubMed Scopus (473) Google Scholar). The AOS production and lipid peroxidation induced by cryptogein are in close correlation with the intensity of necrosis (17Rustérucci C. Blein J.-P. Stallaert V. Ducruet J.-M. Pugin A. Ricci P. Plant Physiol. (Rockv.). 1996; 111: 885-891Crossref PubMed Scopus (130) Google Scholar). Applied to detached leaves cryptogein causes total leaf necrosis, and this model appears perfectly suited to biochemical analyses of necrotic associated processes. Molecular insights on the peroxidation regiospecificity and enantioselectivity of PUFAs were expected to discriminate between a free radical mediated process, or a LOX pathway, i.e.nonspecific versus specific peroxidation, respectively. Thus, lipid peroxidation was analyzed in the present work using a previously established hydroxy fatty acid HPLC assay (39Degousée N. Triantaphylidès C. Montillet J.-L. Plant Physiol. (Rockv.). 1994; 104: 945-952Crossref PubMed Scopus (86) Google Scholar) and was further investigated by chiral phase HPLC (40Feussner I. Balkenhohl T.J. Porzel A. Kühn H. Wasternack C. J. Biol. Chem. 1997; 272: 21635-21642Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Our results demonstrated the involvement of an induced 9-LOX-dependent lipid peroxidation pathway using free fatty acids as substrates. Evidence was provided that PUFA hydroperoxides are responsible for tissue necrosis. The activation of the LOX pathway, leading to a massive production of fatty acid hydroperoxides from membrane lipids, appears as being an active process in plant-hypersensitive cell death. The involvement of this pathway to pathogen growth limitation is also discussed. Cryptogein, prepared according to Bonnet et al. (34Bonnet P. Bourdon E. Ponchet M. Blein J.-P. Ricci P. Eur. J. Plant Pathol. 1996; 102: 181-192Crossref Scopus (132) Google Scholar) was provided by M. Ponchet (INRA, Antibes, France). PUFAs and fatty acid standards were purchased from Fluka (Büchs) or Sigma and MeJA from TCI (Interchim, Montluçon, France). The hydroxy fatty acid chromatographic standards have been previously described (41Degousée N. Triantaphylidès C. Starek S. Iacazio G. Martini D. Bladier C. Voisine R. Montillet J.-L. Anal. Biochem. 1995; 224: 524-531Crossref PubMed Scopus (18) Google Scholar). In addition, 15-HEDE, used as an internal standard for HPLC quantification, was prepared from eicosadienoic acid, according to the previously described procedure (42Martini D. Iacazio G. Ferrand D. Buono G. Triantaphylidès C. Biocatalysis. 1994; 11: 47-63Crossref Scopus (35) Google Scholar), and the chemical structure was assessed by 1H and 13C NMR spectroscopy. An enriched fraction containing 16:3 (16:3/18:3/18:2 composition 37/56/7) was prepared from parsley leaf lipid extract, by TLC and galactolipid hydrolysis, as described below. Tobacco plants (N. tabacum var. Petit Havana) were grown for 8 to 9 weeks in a greenhouse, at 100–120 μmol/m2·s light radiance (HQI-BT 400 watts-D OSRAM lamps, München, Germany), with a 14/10 h, 25/20 °C light/dark cycle and 60% relative humidity. Leaves (about 5 g) were selected in the middle of the stem, detached, and treated with 10 μl of an aqueous solution of cryptogein (0.2 μg/μl) or water for control, as described previously (17Rustérucci C. Blein J.-P. Stallaert V. Ducruet J.-M. Pugin A. Ricci P. Plant Physiol. (Rockv.). 1996; 111: 885-891Crossref PubMed Scopus (130) Google Scholar). For MeJA treatment, tobacco plants were placed into an airtight 25-liter chamber for 5 days, and MeJA (5 μl) was applied on a piece of filter paper (43Avdiushko S. Croft K.P.C. Brown G.C. Jackson D.M. Hamilton-Kemp T.R. Hildebrand D. Plant Physiol. (Rockv.). 1995; 109: 1227-1230Crossref PubMed Scopus (67) Google Scholar). Chemicals, in a 0.5% Tween 80 aqueous solution, were infiltrated between two secondary leaf veins, applying the syringe tip to the epidermis of excised leaves. Leaf petioles were then dipped into water and the leaves kept in the dark at room temperature. Necrosis was assessed from changes with time of leaf water content, and expressed as % of initial FW. Free and bonded hydroxy and hydroperoxy fatty acids were analyzed by HPLC as free hydroxy fatty acids, after NaBH4 reduction and hydrolysis. Tobacco leaves (2.5 g) were homogenized in 0.2n NaOH, 5% (w/v) NaBH4, in the presence of the internal reference 15-HEDE (100 nmol/g FW). The samples were frozen and stored at −20 °C. Extraction was carried out, as described previously (39Degousée N. Triantaphylidès C. Montillet J.-L. Plant Physiol. (Rockv.). 1994; 104: 945-952Crossref PubMed Scopus (86) Google Scholar). An aliquot of the extract (50 μl) was submitted to straight phase HPLC (Waters, Millipore, St. Quentin-Yvelines, France) using a Zorbax rx-SIL column (4.6 × 250 mm, 5 μm particle size, Hewlett-Packard, Les Ullis, France), isocratic elution with 70/30/0.25 (v/v/v) hexane/diethyl ether/acetic acid at a flow rate of 1.5 ml/min, and UV detection at 234 nm. Hydroxy fatty acid isomers were identified using standards (41Degousée N. Triantaphylidès C. Starek S. Iacazio G. Martini D. Bladier C. Voisine R. Montillet J.-L. Anal. Biochem. 1995; 224: 524-531Crossref PubMed Scopus (18) Google Scholar). Quantification was performed with reference to 15-HEDE, assuming that all hydroxy fatty acids have the same extinction coefficient at 234 nm. For the enantiomer composition analysis, chromatographic peaks were collected from straight phase HPLC and submitted to chiral phase HPLC, using a Chiralcel OD column (250 × 4.6 mm, 5-μm particle size, Diacel Chemical Industries, Interchim, France), with a mobile phase of 95/5/0.1 (v/v/v) hexane/2-propanol/acetic acid at a flow rate of 1 ml/min, and UV detection at 234 nm (40Feussner I. Balkenhohl T.J. Porzel A. Kühn H. Wasternack C. J. Biol. Chem. 1997; 272: 21635-21642Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). For the analysis of free hydroperoxide and hydroxy fatty acids, tobacco leaf tissue (2.5 g) was ground at 4 °C in 6 ml of 100 mmpotassium phosphate buffer, pH 4.5, containing 1 mm both deferoxamine mesylate and ortho-phenanthroline, as transition metal chelators, and 10 ml of 50/50 (v/v) chloroform/methanol with 2 mm triphenyl phosphine, as reducing agent, and in the presence of 15-HEDE (100 nmol/g FW). The organic phase was recovered by centrifugation at 700 ×g for 5 min, and the aqueous phase was further extracted with 5 ml of chloroform. Both organic phases were pooled before vacuum evaporation of the solvent without drying. Then, 2 × 10 ml of 70/30 (v/v) hexane/ethyl ether was added and evaporated again to eliminate chloroform. The residue was applied onto a silica Sep-Pak cartridge column (Millipore, St. Quentin-Yvelines, France), previously equilibrated with 70/30 (v/v) hexane/ethyl ether. Product elution was carried out successively with 1 ml of 70/30/0.1 (v/v/v) hexane/ethyl ether/acetic acid and 10 ml of 70/30/1 (v/v/v) hexane/ethyl ether/acetic acid. The first 1 ml was discarded and the next volume recovered, evaporated to 800 μl, and analyzed by straight phase HPLC as described above. In order to assess for free fatty acid hydroperoxides, a second extraction of the same sample was carried out in parallel in which triphenyl phosphine was omitted in the extraction buffer. The extraction procedure and HPLC analysis was the same as above and led to the separation of both free hydroperoxide and hydroxy fatty acids. The overall fatty acid composition of tobacco leaf sample (80 mg FW) was determined according to Miquel and Browse (44Miquel M. Browse J. J. Biol. Chem. 1992; 267: 1502-1509Abstract Full Text PDF PubMed Google Scholar). Fatty acid methyl esters were analyzed by gas phase chromatography (Hewlett-Packard 5890 series II, Les Ullis, France) on a 15-meter × 0.53-mm Carbowax column (Alltech Associates, Deerfield, IL) with flame ionization detection. The oven temperature was programmed for 1 min at 160 °C, followed by a 20 °C/min ramp to 190 °C and a second ramp of 5 °C/min to 210 °C, and maintained at 210 °C for a further 5 min. The methyl ester fatty acid peaks were quantified and identified by comparison of their retention times with those of standards. Data are expressed in nmol/mg DW. For individual lipid analysis, tobacco leaves (0.5 g) were ground under liquid nitrogen using a mortar and pestle. The tissue sample was transferred into a screw-capped centrifuge tube with 6 ml of 10/10/1 (v/v/v) chloroform/methanol/formic acid and stored overnight at −20 °C. After centrifugation (2,000 × g, 5 min), the supernatant was collected and the tissue pellet re-extracted with 2.2 ml of 5/5/1 (v/v/v) chloroform/methanol/water. Both extracts were combined and washed with 3 ml of 0.2 mH3PO4, 1 m KCl. Lipids were recovered in the chloroform phase, dried under N2, and dissolved in 0.5 ml of 2/1 (v/v) chloroform/methanol. Individual lipids were purified from the extracts by monodimensional TLC using either 25/25/25/10/9 (v/v/v/v/v) chloroform/methyl acetate/n-propyl alcohol/0.25% aqueous KCl (w/v) for polar lipids or 90/15/2 (v/v/v) hexane/diethyl ether/acetic acid for neutral lipids. Lipids were then located by spraying the plates with a solution of 0.001% (w/v) primuline in 80% acetone, followed by visualization under UV light. The silica gel zones corresponding to individual lipids were scraped from the plates, and fatty acid methyl esters were prepared and analyzed as described above. Frozen leaf tissue (2 g) was ground in ice-cold 100 mm, pH 6, sodium phosphate buffer (4 ml), containing 2% (w/v) of polyvinyl polypyrrolidone, and a protease inhibitor mixture (½-tablet for 4 ml of buffer, Roche Molecular Biochemicals CompleteTM). The mixture was centrifuged for 20 min at 16,000 × g, and the pellet was discarded. This crude extract was used for the LOX assay and protein quantification. The enzyme extract (0.5 ml) was incubated for 20 min at 25 °C, with 0.25 m sodium phosphate buffer, pH 7, at a final volume of 1.5 ml, and 5 μl of 18:2 (0.1 m) in ethanol. The reaction was stopped by adding 200 μl of 1n NaOH and 500 μl of 5% (w/v) NaBH4 in 0.2n NaOH. After addition of the internal reference (100 nmol of 15-HEDE), hydroxy fatty acids were extracted in 1.5 ml of 70/30 (v/v) hexane/diethyl ether and analyzed by straight phase HPLC and chiral phase as above. Protein content was determined using Pierce BCA protein assay reagent, following the enhanced protocol of the manufacturer's instructions (Pierce), with bovine serum albumin as standard. Total RNA fraction was extracted from control and treated leaves at various times. RNA extraction was performed using the Plant RNA easy minikit, and poly(A)+ RNA extraction was carried out using an Oligotex mRNA kit (both from Qiagen, Courtaboeuf, France) following the manufacturer's instructions. The LOX-specific probe was prepared by RACE amplification using the MarathonTM cDNA Amplification Kit (CLONTECH, Ozyme, St. Quentin-Yvelines, France) with 1 μg of poly(A)+ RNA extracted from tobacco cells treated with cryptogein for 60 min. The two primers used for the 5′-RACE reactions were a gene-specific primer deduced from the sequence of the LOX1 of N. tabacum (45Véronési C. Fournier J. Rickauer M. Marolda M. Esquerré-Tugayé M.-T. Plant Physiol. (Rockv.). 1995; 108: 1342Google Scholar) (GenBankTMaccession number X84040), 5′-GAGGAGTAGCTGTTGAGGACTGGAGCTCCC-3′ (30-mers), and a primer corresponding to the Marathon adapter 5′-CCATCCTAATACGACTCACTATAGGGC-3′ (27-mers). Briefly, poly(A)+ RNA were reverse-transcribed with Moloney murine leukemia virus-reverse transcriptase using (T)30-NN as primer. The second strand performed with a mixture of Escherichia coliDNA polymerase I, RNase H, and E. coli DNA ligase was monitored by addition of [32P]dCTP. Following the creation of blunt ends with T4 DNA polymerase, the double strand cDNA was ligated to the Marathon cDNA-Adapter. The 5′-RACE reaction was performed on this cDNA population with the ExpandTM Long Template PCR System (Roche Molecular Biochemicals) for 30 cycles using the following steps: 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 4 min. The PCR products were analyzed by electrophoresis on 1.2% agarose gel in TAE buffer. One unique band of almost 1 kilobase pair was extracted from the gel and cloned in pGEM-Teasy vector (Promega, Charbonnieres, France). Fluorescent sequencing was done by Genome Express S.A. (Grenoble, France) using SP6 as the downstream primer and a custom-designed primer as the upstream primer. Sequence analysis were carried out with FASTA, NCBI, and the Wisconsin Sequence Analysis Package (Genetics Computer Group, WI). The obtained sequence (998 base pairs) showed 98% identity with the tobacco LOX1. This cDNA was used as specific LOX probe for RNA analysis. Northern blots were carried out according to standard protocols using 15 μg of total RNA per lane. After electrophoresis, RNA samples were transferred and UV cross-linked to Hybond N+ filters (Amersham Pharmacia Biotech). Hybridization was carried out with the specific LOX cDNA, 32P-labeled by random priming (rediprime, Amersham Pharmacia Biotech), at 42 °C overnight as described previously (33Suty L. Petitot A.-S. Lecourieux D. Blein J.-P. Pugin A. Plant Physiol. Biochem. 1996; 34: 443-451Google Scholar). Filters were washed with 2 × SSC, 0.1% SDS at room temperature, 4 × 10 min, then with 0.2 × SSC, 0.1% SDS at 60 °C 2 × 10 min, and analyzed with a PhosphorImager (Molecular Dynamics, Les Bordes, France). Lipid peroxidation was investigated in tobacco leaves using straight phase HPLC analysis of hydroxy fatty acids obtained after NaBH4 reductive extraction and NaOH hydrolysis of total lipids (39Degousée N. Triantaphylidès C. Montillet J.-L. Plant Physiol. (Rockv.). 1994; 104: 945-952Crossref PubMed Scopus (86) Google Scholar). Typical chromatograms of hydroxy fatty acids extracted from control leaves and cryptogein-treated leaves, when necrotic areas appeared, are described in Fig. 1 A. All positional isomers of 18:2 and 18:3 hydroxy fatty acids were separated and the chromatographic peaks attributed. From our experiments, 16:3 represents in tobacco leaves about 15% of total fatty acids (see below), and the occurrence of corresponding hydroxy fatty acids was investigated. Two isomers were enzymatically prepared and identified (see legend of Fig. 1), but they were not detected either in control or elicited leaf extracts. Therefore the 16:3 hydroxy fatty acids were not investigated further. In control leaves, the level of hydroxy fatty acids is low with a preeminence for 13-HODE and 13-HOTE. In cryptogein-treated leaves, lipid peroxidation increased markedly, as observed on all 18:2 and 18:3 positional isomers, the major isomers being 9-HODE and 9-HOTE. Since 9- or 13-LOX were characterized in plants, positional 9 and 13 isomers can arise from either LOX activity or autoxidation, whereas 12-HOTE and 16-HOTE can be considered to be specific of fatty acid autoxidation. In addition, if peroxidation products are chiral, they necessarily arise from LOX activity, whereas racemic products can be obtained either by autoxidation or by a nonspecific LOX. Each chromatographic peak was collected and submitted to chiral phase HPLC, the example of (9R)- and (9S)-HOTE enantiomer separation in control and treated samples being described in Fig. 1,B and C. All the isomers were racemic mixtures in control leaves, with the exception of 13-HODE and 13-HOTE which exhibited an (S)/(R) enantiomeric ratio of 80/20 and 90/10, respectively (Table I). In elicited leaves, as expected, 12- and 16-HOTE remained racemic, 13-HODE and 13-HOTE (S)/(R) enantiomeric ratios were unchanged, whereas 9-HODE and 9-HOTE (S)/(R) enantiomeric ratios reached values around 90/10 (Table I).Table IEnantiomer composition of the hydroxy fatty acids obtained by the NaBH 4 /hydrolysis procedure from control and cry" @default.
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W2108372224 date "1999-12-01" @default.
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W2108372224 title "Involvement of Lipoxygenase-dependent Production of Fatty Acid Hydroperoxides in the Development of the Hypersensitive Cell Death induced by Cryptogein on Tobacco Leaves" @default.
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W2108372224 doi "https://doi.org/10.1074/jbc.274.51.36446" @default.
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