Involvement of lipoxygenase-dependent production of fatty acid hydroperoxides in the development of the hypersensitive cell death induced by cryptogein on tobacco leaves

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Involvement of lipoxygenase-dependent production of

fatty acid hydroperoxides in the development of the

hypersensitive cell death induced by cryptogein on

tobacco leaves

Christine Rusterucci, Jean-Luc Montillet, Jean-Pierre Agnel, Christine

Battesti, Béatrice Alonso, Anja Knoll, Jean-Jacques Bessoule, Philippe

Etienne, Lydie Suty, J Pierre Blein, et al.

To cite this version:

Christine Rusterucci, Jean-Luc Montillet, Jean-Pierre Agnel, Christine Battesti, Béatrice Alonso, et

al.. Involvement of lipoxygenase-dependent production of fatty acid hydroperoxides in the

develop-ment of the hypersensitive cell death induced by cryptogein on tobacco leaves. Journal of Biological

Chemistry, American Society for Biochemistry and Molecular Biology, 1999, 274 (51), pp.36446-36455.

�10.1074/jbc.274.51.36446�. �hal-02696304�

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Involvement of Lipoxygenase-dependent Production of Fatty Acid

Hydroperoxides in the Development of the Hypersensitive Cell

Death induced by Cryptogein on Tobacco Leaves*

(Received for publication, July 21, 1999, and in revised form, September 22, 1999)

Christine Ruste´rucci‡, Jean-Luc Montillet‡, Jean-Pierre Agnel‡, Christine Battesti‡,

Be´atrice Alonso‡, Anja Knoll§, Jean-Jacques Bessoule§, Philippe Etienne¶_{, Lydie Suty}¶_,

Jean-Pierre Blein¶, and Christian Triantaphylide`s‡i

From the ‡CEA-Cadarache, Direction des Sciences du Vivant, De´partement d’Ecophysiologie Ve´ge´tale et de Microbiologie, Laboratoire de Radiobiologie Ve´ge´tale, 13108 Saint-Paul Lez Durance Cedex, §Laboratoire de Biogeńe`se Membranaire, CNRS, UMR 5544, Universite´ Victor Se´galan Bordeaux II, 146 Rue Leó Saignant, 33076 Bordeaux Cedex, and¶Unite´ Associeé INRA-Universite´ de Bourgogne 692, Laboratoire de Phytopharmacie et Biochimie des Interactions Cellulaires, BV 1540, Dijon Cedex, France

Lipid peroxidation was investigated in relation with the hypersensitive reaction in cryptogein-elicited to-bacco leaves. A massive production of free polyunsatu-rated 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 con-comitant increase of 9-LOX activity, and preceded by a transient accumulation of LOX transcripts. Free radi-cal-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 evi-dence 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.

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 de-pendent on a gene for gene plant pathogen interaction (1, 2). HR is accompanied by a battery of defense mechanisms includ-ing de novo synthesis of antimicrobial enzymes and metabo-lites, strengthening of the cell wall, and the onset of systemic acquired resistance dependent on salicylic acid accumulation (3, 4). HR often leads to dry lesions that are supposed to limit pathogen growth. Other proposed roles is the release in ap-oplasm 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 (1, 2). Furthermore, hypersensitive cell death has morphologi-cal and molecular features similar to the mammalian PCD, called apoptosis. These include cytoplasm and chromatin con-densation followed by their fragmentation, activation of calcium-dependent endonucleases (5– 8) and of cysteine proteases (9 – 11), and involvement of similar regulation factors (2). Some differences between HR and mammalian apoptosis were ob-served, however, such as changes in DNA laddering (5, 8) and the lack in HR of the repressor role of Bcl-xL(12). One ultimate characteristic of HR is the loss of membrane integrity, and thus HR is often characterized by an associated electrolyte leakage (5, 13). This feature is not encountered in mammalian apopto-sis but is one characteristic of the catastrophic cell death called necrosis, which is not dependent on gene activation (14). In this way, the use of the term “necrosis” assumes different meanings when referring to mammalian cell death or to pathogen-asso-ciated plant cell death.

Membrane damage during HR is in close correlation with lipid peroxide production and with AOS generation (12, 15–17). AOS can initiate lipid peroxidation in membranes by fatty acid free radical production, and the process can be propagated by autoxidation (18). The generation of AOS during the oxidative

* This work was supported by European Community funding, as a part of the “CAST” project within the 4th Framework Program in Biotechnology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

iTo whom correspondence should be addressed: CEA, Laboratoire de Radiobiologie Ve´ge´tale, De´pt. d’Ecophysiologie Ve´ge´tale et de Microbi-ologie, Centre d’Etudes de Cadarache, 13108 Saint-Paul Lez Durance Cedex, France. Tel.: 33 (0)4 42 25 64 86; Fax: 33 (0)4 42 25 62 86; E-mail: ctriantaphylid@cea.fr.

1_{The abbreviations used are: HR, hypersensitive reaction; PCD,}

pro-grammed cell death; AOS, active oxygen species; LOX, lipoxygenase; PUFA, polyunsaturated fatty acid; HPLC, high pressure liquid chromatography; JA, jasmonate; MeJA, methyl jasmonate; 15-HEDE, 15-hydroxy-11,13(Z,E)-eicosadienoic acid; FW, fresh weight; DW, dry weight; RACE, rapid ampli-fication of cDNA ends; PCR, polymerase chain reaction; 16.0, palmitic acid; 16:1, palmitoleic acid; t-16:1, trans-palmitoleic acid; 16:2, hexadecadienoic acid; 16:3, hexadecatrienoic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, linolenic acid; 9-HODE, 9-hydroxy-10,12(Z,E)-octadecadi-enoic acid; t-9-HODE, 9-hydroxy-10,12 (E,E) octadecadi9-hydroxy-10,12(Z,E)-octadecadi-enoic acid; 13-HODE, 9,11(Z,E)-octadecadienoic acid; t-13-13-HODE, 13-hydroxy-9,11(E,E) octadecadienoic acid; 9-HOTE, 9-hydroxy-10,12,15(E,Z,Z)

octa-decatrienoic acid; 12-HOTE, 12-hydroxy-9,13,15,(Z,E,Z)-octaocta-decatrienoic acid; 13-HOTE, 13-hydroxy-9,11,15(Z,E,Z)-octadecatrienoic acid; 16-HOTE, 16-hydroxy-9,12,14,(Z,Z,E)-octadecatrienoic acid; 9-HPODE, 9-hydroper-oxy-10,12(Z,E)-octadecadienoic acid; 13-HPODE, 13-hydroperoxy-9,11(Z,E)-octadecadienoic acid; 9-HPOTE, 9-hydroperoxy-10,12,15(E,Z,Z) octadecatrienoic acid; 13-HPOTE, 13-hydroperoxy-9,11,15(Z,E,Z)-octadecatrienoic acid; tert-BuOOH, tert-butyl hydroperoxide.

This paper is available on line at http://www.jbc.org

36446

at INRA Institut National de la Recherche Agronomique, on September 16, 2010

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burst is an important early event during the course of plant-pathogen interactions and is well documented (19, 20). 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 per-oxidation and hypersensitive cell death (15, 21–23).

Lipid peroxidation might also be due to LOX (EC 1.13.11.12) activity (24, 25). 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 (26, 27). Indeed, induction of LOX activity has been observed in several plants during incompatible interaction and occurs after a lag phase of few hours (28). The observation that an incom-patible interaction can be suppressed in transgenic tobacco plants expressing antisense LOX clearly demonstrates the role of LOX in plant resistance to pathogens (29). Finally, since (i) the LOX pathway leads to products, such as hydroperoxides, alkenals, and aldehydes, that may kill plant cells and pathogen (30, 31) 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 (32), was investigated in this work. Cryptogein leads

also to defense gene activation (33) and systemic acquired resistance (34). Features of PCD were observed, as assessed by plasma membrane blebbing, cell shrinkage, and cytoplasmic condensation (6), and the expression of hsr 203J, a gene pro-posed as the hallmark of HR-inducing pathogens or elicitors (35). On tobacco cells cryptogein induces an early oxidative burst (36) and late LOX activity (37). An early AOS production was also characterized on leaves (38). The AOS production and lipid peroxidation induced by cryptogein are in close correlation with the intensity of necrosis (17). 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 regiospecific-ity and enantioselectivregiospecific-ity of PUFAs were expected to discrim-inate between a free radical mediated process, or a LOX path-way, 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 (39) and was further investigated by chiral phase HPLC (40). 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 hy-droperoxides 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.

EXPERIMENTAL PROCEDURES

Chemicals—Cryptogein, prepared according to Bonnet et al. (34) was

provided by M. Ponchet (INRA, Antibes, France). PUFAs and fatty acid standards were purchased from Fluka (Bu¨chs) or Sigma and MeJA from TCI (Interchim, Montluc¸on, France). The hydroxy fatty acid chromato-graphic standards have been previously described (41). In addition,

15-HEDE, used as an internal standard for HPLC quantification, was prepared from eicosadienoic acid, according to the previously described procedure (42), and the chemical structure was assessed by1_{H and}13_C

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.

Plant Growth and Treatments—Tobacco plants (N. tabacum var.

Petit Havana) were grown for 8 to 9 weeks in a greenhouse, at 100 –120 mmol/m2_{zs light radiance (HQI-BT 400 watts-D OSRAM lamps,}

Mu¨ nchen, 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 10ml of an aqueous solution of cryp-togein (0.2mg/ml) or water for control, as described previously (17). For MeJA treatment, tobacco plants were placed into an airtight 25-liter chamber for 5 days, and MeJA (5ml) was applied on a piece of filter paper (43). 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 ex-pressed as % of initial FW.

Hydroxy Fatty Acid and Hydroperoxy Fatty Acid Analysis—Free and

bonded hydroxy and hydroperoxy fatty acids were analyzed by HPLC as free hydroxy fatty acids, after NaBH4reduction and hydrolysis. Tobacco

leaves (2.5 g) were homogenized in 0.2NNaOH, 5% (w/v) NaBH4, in the

presence of the internal reference 15-HEDE (100 nmol/g FW). The samples were frozen and stored at220 °C. Extraction was carried out, as described previously (39). An aliquot of the extract (50 ml) was submitted to straight phase HPLC (Waters, Millipore, St. Quentin-Yvelines, France) using a Zorbax rx-SIL column (4.63 250 mm, 5mm 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 (41). 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 (2503 4.6 mm, 5-mm 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 (40).

For the analysis of free hydroperoxide and hydroxy fatty acids, to-bacco leaf tissue (2.5 g) was ground at 4 °C in 6 ml of 100 mMpotassium phosphate buffer, pH 4.5, containing 1 mMboth deferoxamine mesylate and ortho-phenanthroline, as transition metal chelators, and 10 ml of 50/50 (v/v) chloroform/methanol with 2 mMtriphenyl phosphine, as reducing agent, and in the presence of 15-HEDE (100 nmol/g FW). The organic phase was recovered by centrifugation at 7003 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, 23 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 800ml, 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.

Fatty Acid and Lipid Analysis—The overall fatty acid composition of

tobacco leaf sample (80 mg FW) was determined according to Miquel and Browse (44). Fatty acid methyl esters were analyzed by gas phase chromatography (Hewlett-Packard 5890 series II, Les Ullis, France) on a 15-meter3 0.53-mm Carbowax column (Alltech Associates, Deerfield, IL) with flame ionization detection. The oven temperature was pro-grammed 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 stand-ards. 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

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transferred into a screw-capped centrifuge tube with 6 ml of 10/10/1 (v/v/v) chloroform/methanol/formic acid and stored overnight at220 °C. After centrifugation (2,0003 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 0.2MH3PO4, 1MKCl. 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.

LOX Activity Determination—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 (1_⁄₂_{-tablet for 4 ml of buffer, Roche Molecular Biochemicals}

Com-pleteTM_{). The mixture was centrifuged for 20 min at 16,000}_{3 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 0.25Msodium phosphate buffer, pH 7, at a final volume of 1.5 ml, and 5ml of 18:2 (0.1M) in ethanol. The reaction was stopped by adding 200ml of 1NNaOH and 500ml of 5% (w/v) NaBH4in 0.2NNaOH. 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.

Preparation of the LOX-specific Probe and RNA Analysis—Total

RNA fraction was extracted from control and treated leaves at various times. RNA extraction was performed using the Plant RNA easy mini-kit, and poly(A)1_{RNA extraction was carried out using an Oligotex}

mRNA kit (both from Qiagen, Courtaboeuf, France) following the man-ufacturer’s instructions. The LOX-specific probe was prepared by RACE amplification using the MarathonTM

cDNA Amplification Kit (CLON-TECH, Ozyme, St. Quentin-Yvelines, France) with 1 mg of poly(A)1

RNA extracted from tobacco cells treated with cryptogein for 60 min. The two primers used for the 59-RACE reactions were a gene-specific primer deduced from the sequence of the LOX1 of N. tabacum (45) (GenBankTM _{accession number X84040), 5}

9-GAGGAGTAGCTGTT-GAGGACTGGAGCTCCC-39 (30-mers), and a primer corresponding to the Marathon adapter 59-CCATCCTAATACGACTCACTATAGGGC-39 (27-mers). Briefly, poly(A)1RNA were reverse-transcribed with Molo-ney murine leukemia virus-reverse transcriptase using (T)30-NN as primer. The second strand performed with a mixture of Escherichia coli DNA polymerase I, RNase H, and E. coli DNA ligase was monitored by addition of [32_{P]dCTP. Following the creation of blunt ends with T4}

DNA polymerase, the double strand cDNA was ligated to the Marathon cDNA-Adapter. The 59-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). Flu-orescent 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 15mg of total RNA per lane. After electrophoresis, RNA samples were transferred and UV cross-linked to Hybond N1_{filters (Amersham}

Pharmacia Biotech). Hybridization was carried out with the specific LOX cDNA, 32

P-labeled by random priming (rediprime, Amersham Pharmacia Biotech), at 42 °C overnight as described previously (33). Filters were washed with 23 SSC, 0.1% SDS at room temperature, 4 3 10 min, then with 0.23 SSC, 0.1% SDS at 60 °C 2 3 10 min, and analyzed with a PhosphorImager (Molecular Dynamics, Les Bordes, France).

RESULTS

LOX-mediated Lipid Peroxidation in Cryptogein-induced HR—Lipid peroxidation was investigated in tobacco leaves

us-ing straight phase HPLC analysis of hydroxy fatty acids ob-tained after NaBH4reductive extraction and NaOH hydrolysis

of total lipids (39). Typical chromatograms of hydroxy fatty acids extracted from control leaves and cryptogein-treated leaves, when necrotic areas appeared, are described in Fig. 1A. 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 correspond-ing 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 ob-served on all 18:2 and 18:3 positional isomers, the major iso-mers being 9-HODE and 9-HOTE. Since 9- or 13-LOX were

FIG. 1. HPLC analysis of hydroxy fatty acids extracted from control and cryptogein-treated leaves after NaBH4reduction

and lipid hydrolysis. Control and cryptogein-treated tobacco leaves were kept in the dark, and after 24 h, total hydroxy acids from free and bonded fatty acids were extracted, using the NaBH4

reduction-hydrol-ysis procedure and submitted to straight phase HPLC, as described under “Experimental Procedures.” A, HPLC traces of cryptogein-treated leaf extract (lower trace) and of control leaf extract (upper trace), using a Zorbax rx-SIL column. The various hydroxy fatty acids isomers of 18:2 and 18:3 fatty acids were identified, as described previously (41), and quantified with reference to the internal standard 15-HEDE. In addition, among the four isomers of 16:3, two were prepared from partly purified 16:3, using soybean and tobacco LOX activities (13- and 9-spe-cific on 18:2, respectively), and were not detected in the chromatograms of both control and elicited leaves (respective retention times 21.1 and 30.9 min). In the chromatogram of cryptogein-treated leaf extract, two early eluting compounds appeared just before 15-HEDE, and were designated X and Y. According to their UV spectra, both compounds were not identified as hydroxy fatty acids. Hydroxy fatty acids were collected from the preceding HPLC analyses and submitted to chiral phase HPLC, using a Chiralcel OD column, as described under “Exper-imental Procedures.” B, separation of the (9R)-HOTE and (9S)-HOTE enantiomers from control leaf extract. C, separation of the (9R)-HOTE and (9S)-HOTE enantiomers from cryptogein-treated leaf extract.

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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 non-specific 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 sam-ples 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 un-changed, whereas 9-HODE and 9-HOTE (S)/(R) enantiomeric ratios reached values around 90/10 (Table I).

These data suggested that lipid peroxidation in elicited leaves is dependent on (i) an intense LOX metabolism and (ii) a weak autoxidation pathway, which was observed through the increased levels of the non-enzymatic products 12- and 16-HOTE. In control leaves, metabolites of the LOX pathway were observed at a low level and presented 13S-specificity. In elic-ited leaves, both 13- and 9-isomer levels increased and pre-sented S-enantioselectivity. Thus, the time course of lipid per-oxidation was analyzed in Fig. 2A in terms of LOX-dependent peroxidation, illustrated by the evolution level of the 13-iso-mers on the one hand and of the 9-iso13-iso-mers on the other hand, and autoxidation, illustrated by the increase in the level of 12-HOTE1 16-HOTE. Both types of enzymatic and non-enzy-matic peroxidation appeared to be induced simultaneously af-ter a 12-h lag phase. For the initial constitutive 13-specific LOX-dependent peroxidation, the metabolite level was shown to increase, from 0.04mmol/g FW reaching a plateau at 0.18 mmol/g FW after 15 h. The level of the metabolites at the 9-position increased markedly and continuously reaching 0.9 mmol/g FW after 30 h. The differences in kinetics of 13- and 9-isomer accumulation suggest the involvement of at least two different LOXs with 13S- and 9S-specificity, respectively. Glo-bally, LOX-dependent peroxidation appeared to be about 10 times greater than autoxidation. As expected from a previous study that used the thiobarbituric assay (17), the level of lipid peroxidation was shown to be correlated to cryptogein-induced leaf necrosis, as measured by tissue dehydration (Fig. 2B).

Time Course of LOX Induction in Cryptogein-elicited Leaves—The steady-state level of mRNA encoding for LOX was

analyzed using Northern blot experiments at different times following cryptogein application to tobacco leaves. A specific probe corresponding to tobacco LOX 1, described by Ve´rone´si et

al. (45), was prepared by RACE-PCR and recognized a

2.9-kilobase pair mRNA band. The accumulation of LOX mRNAs

monitored in excised tobacco leaves infiltrated with cryptogein or with water is described in Fig. 3, A and B. A high transitory accumulation occurred between 6 and 8 h after the treatment with cryptogein, whereas no accumulation was observed in the water-treated leaves. Changes in 9-LOX activity were analyzed in the same experiment and are described in Fig. 3C. In addi-tion, in both elicited and control leaves, attempts to character-ize 13-LOX activity, as suggested by the previous metabolite analyses, were not successful (see below). In elicited leaves, the time course of 9-LOX activity level followed a similar transitory increase, reaching an apparent maximum (27 picokatals/mg protein) after 16 h after a lag phase of 8 h. An important decrease was then observed (7 picokatals/mg protein after 28 h). In control leaves the activity was at the limit of the detection level (,2 picokatals/mg protein) for at least 72 h.

Product Specificity and Substrates of Elicited

LOX—Analy-ses of LOX metabolites were conducted to asLOX—Analy-sess free fatty acid hydroperoxide formation by cryptogein action. Since the NaBH4/hydrolysis extraction procedure leads to the global

analysis of free and esterified hydroperoxide and hydroxy fatty acids, in a first type of experiment, reductive conditions were designed, in which the hydrolytic step was omitted. Lipids were extracted from 24-h cryptogein-treated leaves, by grinding at pH 4.5, in the presence of iron chelators to avoid hydroperoxide

TABLE I

Enantiomer composition of the hydroxy fatty acids obtained by the NaBH4/hydrolysis procedure from control and cryptogein-treated

leaves

Hydroxy fatty acids were obtained and analyzed according to the procedure described in Fig. 1. Results are expressed as mean6 S.D. of three independent analyses.

Hydroxy fatty acids

Metabolite enantioselectivity S/R ratio Control, 0 h Control, 24 h Cryptogein,_{24 h}

9-HODE 51/496 5 51/496 8 88/126 2 9-HOTE 49/516 3 48/526 1 91/096 1 13-HODE 80/206 7 79/216 5 72/286 2 13-HOTE 90/106 6 87/136 6 84/166 1 12-HOTE 53/476 4 53/476 5 57/436 3 16-HOTE 54/466 4 51/496 5 54/466 2

FIG. 2. Time course of LOX-dependent peroxidation and au-toxidation in control and cryptogein-treated leaves; relation-ship with tissue necrosis. Hydroxy fatty acids were extracted from control and cryptogein-treated leaves at different times following appli-cation, using the NaBH4reduction-hydrolysis procedure and analyzed

by HPLC as described in Fig. 1. A, open and closed symbols represented the level of hydroxy fatty acids for water-treated and cryptogein-treated leaves, respectively. Œ, total hydroxy fatty acids; ●, 9-HOTE1 9-HODE representative of 9-LOX activity; f, 13-HODE1 13-HOTE represent-ative of 13-LOX activity; inset,l, 12-HOTE 1 16-HOTE, representa-tive of PUFA autoxidation. B, relationship between leaf necrosis and total lipid peroxidation; tissue necrosis was evaluated by measurement of leaf dehydration and total lipid peroxidation by total hydroxy fatty acid level. Mean and S.D. from three independent experiments are given.

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degradation, and with triphenyl phosphine in the chloroform/ methanol mixture to reduce the hydroperoxides under mild conditions (42). Although HPLC of these extracts appeared to be more complex, as compared with the NaBH₄/hydrolysis pro-cedure, free hydroxy fatty acids could, however, be analyzed by straight phase HPLC, after a silica Sep-Pak column enrich-ment. Under such experimental conditions, only the free 9-hy-droxy fatty acid isomers were observed, and the 9-HODE/9-HOTE ratio was 20/80 (results not shown). The 9-hydroxy fatty acids were subsequently collected, submitted to chiral phase HPLC, and shown to present the expected 90% S-enantioselec-tivity. The free 9-hydroxy fatty acids recovered under these conditions represented 606 18% (n 5 4) of the total 9-hydroxy fatty acids obtained by the NaBH4/hydrolysis extraction

proce-dure. In a second type of experiment, the triphenyl phosphine was not present in the extraction buffer. Free fatty acid hy-droperoxides as well as free hydroxy fatty acids could then be identified and quantified by straight phase HPLC analysis. The HPLC analyses of free fatty acid hydroperoxides described in Fig. 4 clearly show that, among the various possible positional hydroperoxide isomers, only 9-HPODE and 9-HPOTE were observed and, as above for free 9-hydroxy fatty acids, in a 20/80 ratio. By using the latter extraction procedure, the free 9-hy-droperoxides represented 51_{6 7% (n 5 2) of the sum of free} 9-hydroperoxide and 9-hydroxy fatty acids.

The preceding results suggested that free PUFAs should be the substrates of cryptogein-induced LOX. Thus, LOX activity was measured using 18:2 as a substrate and by HPLC to assess for 9/13 specificity. The activity showed a maximum value at pH 7. Cryptogein-induced LOX was shown as being

regio-spe-cific producing 98% of 9-HODE, and the speregio-spe-cificity was similar between pH 5 and 9. Substrate specificity was analyzed by competition in a mixture of 16:3/18:3/18:2 (37/56/7, respective-ly). All the PUFAs were substrates and shown to be peroxidized with relative reaction rates of 16/79/5, for 16:3/18:3/18:2, re-spectively. In addition, since MeJA has been described to in-duce transcriptional activation of the 9-LOX gene and accumu-lation of 9-LOX activity in tobacco (46), the MeJA-induced LOX was analyzed similarly. Tobacco plants were treated with MeJA for 5 days, under the conditions described by Avdiushko

et al. (43). The LOX activity analyzed on leaves was shown to be

at the same level as in leaves treated with cryptogein for 16 h and to exert the same regio-specificity, 9-HODE representing over 96% of the products. The enantioselectivity of MeJA- and cryptogein-induced LOXs was determined on 9-HODE produc-tion, either in vitro, with an enzymatic extract, or in planta, after 18:2 substrate infiltration into leaf and metabolite anal-ysis (Table II). Both enzymatic extracts possessed the same 9S-enantioselectivity (around 92%). The 18:2 infiltration ex-periments led to similar increased levels of (9S)-HODE and similar enantioselectivity (around 86%). In both cases, al-though the induced LOX appeared very specific at the 9-posi-tion, the infiltration experiments led, in addi9-posi-tion, to increased levels of (13S)-HODE (around 85% enantioselectivity; Table II). This result suggested again the presence of a (13S)-LOX, which could not be characterized in vitro in the present experiments.

PUFA Consumption in Cryptogein-elicited Leaves—To

inves-tigate the potential source of free fatty acids as substrates for the induced LOX, global changes in the fatty acid composition of control and elicited leaves were compared after treatment, when the symptoms were not fully developed (18 h), and also compared with the composition of control leaves at the begin-ning of the treatment (Fig. 5A). First, the fatty acid composition of control leaves remained unchanged after leaf excision. Sec-ond, the composition of all fatty acids did not change signifi-cantly in elicited leaves with the exception of 18:2 and 18:3, the major fatty acid in leaves, which decreased to 636 8% and 70 6 9% (n_{5 3) of initial levels, respectively. The different classes of} lipids were analyzed for their fatty acid composition, and the decrease was confirmed for 18:2 and 18:3. As shown in Fig. 5B,

FIG. 3. Time course of the 9-LOX gene expression and of 9-LOX

activity in control and cryptogein-treated leaves. Tobacco leaves

were harvested after various times and frozen in liquid nitrogen. Total RNA or protein extraction was carried out as described under “Exper-imental Procedures.” A, Northern blot analysis of LOX transcripts accumulating in cryptogein-treated tobacco leaves (15mg of total RNA per lane); upper panel, hybridization with the tobacco LOX probe; lower

panel, ethidium bromide staining of rRNA, as loading control. B,

North-ern blot analysis of LOX transcripts from water-treated leaves; condi-tions as in A. C, evolution of 9-LOX activity analyzed in the same preceding experiment, with 18:2 as substrate; open and closed symbols represented the level of 9-LOX activity for water-treated and crypto-gein-treated leaves, respectively. Mean and S.D. from analyses carried out on three independent extracts.

FIG. 4. HPLC characterization of free hydroperoxy fatty acids extracted from cryptogein-treated leaves. Total lipids of 24 h cryp-togein-treated leaves were extracted, omitting reduction, by the meth-anol/chloroform procedure in the presence of iron-chelating compounds, as described under “Experimental Procedures.” Hydroperoxides and hydroxy fatty acids, as well, were collected from a silica Sep-Pak column and subjected to straight phase HPLC, using a Zorbax rx-SIL column.

Trace A, partial chromatogram depicting hydroperoxide separation

from cryptogein-treated leaf extract. Trace B, reference hydroperoxides were prepared from an equimolar mixture of 18:2 and 18:3 (1 mM), with soybean LOX (Fluka; 1 mg/ml) in a pH 7 tetraborate buffer (10 mM), leading to a nonspecific peroxidation (42); after 20 min reaction at 25 °C, hydroperoxides were extracted with hexane/ethyl ether (70/30) and submitted to HPLC, as above; the 13- and 9-isomers, mentioned in

trace B, were collected, reduced with NaBH4, and identified as hydroxy

fatty acids.

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decrease was observed only in phosphatidylcholine and galac-tolipids. Proportional consumption of 18:2 and 18:3 was impor-tant in phosphatidylcholine, reaching 41 6 5 and 74 6 10% (n5 3) of initial level, respectively, and represented a total of 4.6mmol/g DW. Galactolipids appeared quantitatively as the major source of PUFA consumption, since 18:2 and 18:3 reached 576 11 and 61 6 7% (n 5 3) of initial level, respec-tively, and represented a consumption of 1.2 and 20 mmol/g DW, respectively. An increase in the level of free 18:2 and 18:3 was also noticed, whereas the levels of other free fatty acids did not change. Obviously, when symptoms were fully developed (24 h), the total level of all fatty acids started to decrease

significantly (15–20% of consumption), whereas 18:2 and 18:3 reached 53_{6 8 and 54 6 9% (n 5 3) of initial levels,} respec-tively. In accordance with the observation that 16:3 hydroper-oxides do not accumulate in elicited leaves, it is worth men-tioning that the decrease in 16:3 level was low, similar to fatty acids that are not LOX substrates. With reference to the con-trol, the material balance calculation carried out on 18:3 indi-cated that, 24 h after cryptogein treatment, the steady-state level of 9-LOX-dependent peroxidation, as measured by 9-HOTE level according to the NaBH4/hydrolysis procedure,

represented 10 _{6 6% (n 5 3) of 18:3 consumption. Taken} together, these results suggest the involvement of specific lipase(s) in the process and demonstrate the participation of the chloroplastic lipids.

Inhibition of Cryptogein-induced LOX Metabolism and Cell Death by Limiting Oxygen Availability—HR can be inhibited in

the absence of oxygen (12). In a first type of experiment, cryp-togein-treated leaves were kept under normal atmosphere for 1 h, allowing the initiation of the oxidative burst, and then transferred at low oxygen pressure (0.6%) for a further 25 h. As compared with leaves under normal atmosphere, the leaves did not develop the necrotic symptoms. LOX activity was shown to be induced at low oxygen pressure at comparable level as in leaves under normal conditions, but metabolites were not syn-thesized (results not shown). In a second type of experiments, oxygen availability was limited by water-dipping experiments. Cryptogein-treated and control leaves were kept under normal atmosphere for 1 h and then half-dipped into water for the next 23 h in order to compare symptom development on the same leaf. As described in Fig. 6A, necrotic symptoms developed in the upper aerial leaf part of cryptogein-treated leaves but not in the lower immersed part. Although LOX activity was in-duced in both parts at similar levels, metabolite analysis showed increase of lipid peroxidation in the upper part but not in the lower (Fig. 6B). Control leaves did not show any symp-tom or activation of LOX metabolism. In both types of experi-ments, the inhibition of HR was reversed, as soon as leaves were transferred under normal oxygen conditions. These re-sults showed that LOX activity can be induced under low oxygen conditions. However, since oxygen is the PUFA co-substrate of LOX, LOX-dependent peroxidation is inhibited when oxygen is limiting, and consequently cell death.

Activation of Cell Death by LOX Substrates and Metabo-lites—Substrates and products of the LOX pathway were tested

in the induction of tobacco leaf tissue cell death. Although PUFA 9-hydroperoxides could not be prepared with sufficient purity to be tested (47), 13-HPODE can be conveniently pre-pared with soybean LOX (42). The effects of 18:2 and 13-HPODE, at 1, 2, 5, and 10 mMconcentration in 0.5% of a Tween 80 aqueous solution, were first compared by infiltration be-tween two secondary veins of leaves. As shown in Fig. 7A, after 4 h of incubation, 13-HPODE infiltration induced necrotic

ar-TABLE II

Enantioselective peroxidation of 18:2, either by LOX extracts or in planta; comparison of cryptogein- and MeJA-treated leaves

The enantioselective transformation of 18:2 into hydroperoxides was investigated in vitro using enzymatic extracts, and in planta, after 18:2 infiltration into leaves, by the NaBH4/hydrolysis extraction procedure. Results are expressed as mean6 S.D. of three independent experiments.

Cryptogein-induced (16 h) MeJA-induced (5 days)

in Vitroa _{in Planta}b _{in Vitro}a _{in Planta}b

9-HODE (S/R) level increasec _92/8_{6 1} _85/15_{6 1 3 4.0 6 1.1} _93/7_{6 1} _88/12_{6 3 3 4.9 6 1.3}

13-HODE (S/R) level increasec _NDd _83/17_{6 3 3 1.7 6 0.5} _NDd _87/13_{6 2 3 2.8 6 0.9} a_{Leaf protein extract was incubated with 18:2, and after NaBH}

4reduction, the reaction products were separated by straight phase HPLC and

analyzed by chiral phase HPLC, as described under “Experimental Procedures.”

b_{18:2 (5 m}

M) was infiltrated into half-leaf, and metabolites were analyzed in both parts by the NaBH4/hydrolysis procedure, as described under

“Experimental Procedures”; incubation was for 30 min for MeJA-treated plants and 1 h for cryptogein-elicited leaves.

c_{HODE level increase due to 18:2 infiltration, as compared with the non-infiltrated part of the leaf.}

d_{ND, not determined; the 9-LOX specificity was greater than 98 and 96% for cryptogein- and MeJA-induced LOX, respectively.}

FIG. 5. Changes in fatty acid composition of tobacco leaves after cryptogein treatment. Total lipids were extracted from control leaves before treatment and 18 h after water or cryptogein application. Fatty acid composition of total lipids, or of the various classes of lipids, separated by TLC, was evaluated by GPC, as described under “Exper-imental Procedures.” A, total fatty acid composition. B, respective com-position in 18:3 and 18:2 fatty acids, of galactolipids (GL), phosphati-dylcholine (PC), phosphatidylglycerol and ethanolamine (PG-PE), neutral lipids (NL), and free fatty acids (FA). Cross-hatched bars, con-trol, time 0; white bars, concon-trol, time 18 h; black bars, cryptogein treatment, time 18 h. In order to compare, results are expressed in mmol/g DW. Mean and S.D. from three analyses of the same experiment are given, in which fatty acid composition of total lipids (A) and of the various classes of lipids (B) were analyzed simultaneously.

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eas above 2 mM concentration, whereas 18:2 infiltration was

effective only at 10 mM(not shown). In a second phase, necrosis

induction by 5 mMof 13-HPODE was compared on the same

leaf with the effects of the corresponding alcohol (13-HODE),

tert-butyl hydroperoxide (tert-BuOOH) and again of the free

fatty acid (18:2), at the same concentrations. After 4 h, only 13-HPODE infiltration induced the necrotic areas (Fig. 7B). Surprisingly, tert-BuOOH was not effective in developing ne-crotic areas, even at a concentration of 20 mM and after 20 h

(results not shown). The symptoms induced by 2 and 5 mM

13-HPODE infiltration were further observed in water-dipping

experiments (see above) indicating that oxygen was then not necessary to induce the necrotic lesions (results not shown). In leaves of MeJA-treated plants, for which 9-LOX activity accu-mulated (see above), and of the corresponding control, necrotic symptoms were not observed. Infiltration of 18:2 and 18:3 between two secondary leaf veins was carried out at 1, 2, 5, and 10 mMconcentration. Leaf tissue was more sensitive to PUFA

infiltration, as compared with the preceding experiments. In control leaves necrotic areas were induced, after 4 h, for 5 mM

concentration and above (results not shown). In the MeJA-treated material, necrotic lesions appeared within 15 min in the 5–10 mM 18:3-infiltrated parts (results not shown), and

within 4 h for all concentrations of both PUFAs (see Fig. 7C for 18:2).

The above efficient hydroperoxide or PUFA concentrations appear consistent with the decrease of the PUFA level observed in cryptogein-treated leaves. On the one hand, taking into account the volume of compound infiltration (0.75 ml/g FW), a final level of 3.75mmol/g of leaf initial FW can be calculated from a 5 mMproduct infiltration. On the other hand, assuming

that 18:21 18:3 fatty acids represent 65% of total fatty acids in leaf lipids (176 3mmol/g FW; n 5 3), the PUFA decrease in the cryptogein-treated leaves (around 50%, after 24 h) can be esti-mated to be around 5.5mmol/g FW, which is a value close to the preceding one. Taken together, these results confirmed that free PUFAs are the LOX substrates in vivo and demonstrated that application or in planta production of PUFA hydroperox-ides, at relevant physiological levels, induce leaf necrosis.

DISCUSSION

The involvement of a LOX pathway causing the development of tissue necrosis during HR is shown for the first time in this work as follows: (i) free fatty acid hydroperoxides are produced massively in tobacco leaves, in response to cryptogein; (ii) the intensity of leaf necrosis is correlated to the level of fatty acid peroxidation, dependent on cryptogein-induced 9-LOX; (iii) since oxygen is the PUFA co-substrate of LOX, LOX metabo-lism and cell death are both blocked when oxygen availability is limited; (iv) finally, leaf necrosis can be induced either by indirect production of hydroperoxides in planta, infiltrating PUFAs into leaves where LOX activity has been induced by MeJA, or by direct infiltration of physiological relevant levels of fatty acid hydroperoxides.

The production of fatty acid hydroperoxides by a plant LOX was initially described in potato-Phytophthora infestans inter-action, but arachidonate, the LOX substrate, was released from the fungus (48). In our model, it is worth emphasizing that plant lipids are the precursors for hydroperoxide production. Thus, our results showed that cryptogein elicits a plant pro-gram to provide adequate substrates and enzymes for free fatty acid hydroperoxide production leading to cell death. Increasing LOX activity during the development of HR was described for many plants. Enzyme specificity appears to be, however, pendent on the plant model. For instance, 13-LOX was de-scribed in rice (49) and soybean (27), whereas 9-LOX was characterized in tomato (50) and tobacco (Ref. 51 and this work). Since it has been further shown that infiltration of 13-HPODE as well as in planta production of 9-HPODE were able to induce tobacco leaf necrosis, these data taken together suggest that, dependent on the plant species, 13- and (or) 9-hydroperoxide isomers of fatty acids can be produced as po-tent effectors of hypersensitive cell death. Surprisingly, infil-tration in the tobacco leaves of tert-BuOOH up to 20 mM

con-centration did not induce necrotic lesions. Thus, it can be proposed that, in addition to the hydroperoxides, fatty acid hydroperoxide metabolites and degradation products would also play an important role in toxicity. Autoxidation of

esteri-FIG. 6. Effect of water dipping on leaf necrosis and on the

peroxidative metabolism of lipids. Cryptogein-treated and control

leaves were half-dipped into water 1 h after the treatment and left to observe the development of the symptoms. The upper and lower parts of leaves were analyzed at different times to determine the evolution of the 9-LOX activity level and of 9-hydroperoxide metabolite level by the NaBH4 reduction-hydrolysis procedure, as described under

“Experi-mental Procedures.” A, symptoms after 24 h of cryptogein-treated leaf (leaf upper face, left panel; leaf lower face, right panel); leaf necrosis appearing only in the upper aerial part. B, evolution of 9-LOX activity level and of 9-hydroxy fatty acid level; in order to compare upper and lower part of the leaves, metabolite analysis results were expressed in mmol/g DW. B, open and closed symbols represented the levels for control and cryptogein-treated leaves, respectively. ●, 9-LOX activity and 9-hydroxy fatty acid level of the upper aerial leaf part; , 9-LOX activity and 9-hydroxy fatty acids level of the lower dipped leaf part. Mean and S.D. of three independent experiments are given.

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fied fatty acids was also observed and tentatively attributed to membrane lipid peroxidation. This process, representing around 10% of the total lipid peroxidation level, occurs late after elicitation and is induced simultaneously with the enzy-matic process. Therefore, it can be proposed that this additional membrane lipid peroxidation is likely initiated by free fatty acid hydroperoxides produced massively via LOX action, rather than AOS generated early during the oxidative burst (38). Anyway, the hypothesis that massive lipid peroxidation occur-ring in cryptogein-induced HR is the result of free radical production by the oxidative burst can be ruled out. Additional experiments, including the study of other models, are needed, however, to demonstrate the systematic occurrence of the LOX pathway in hypersensitive cell death, and the involvement of AOS as effectors or (and) as signaling components in the process (22, 52, 53).

Analysis of changes in the fatty acid composition of crypto-gein-treated leaves was assessed for the importance of the peroxidative pathway. During the first 18 h, whereas the level of the other fatty acids did not change significantly, about 30% of the 18:2 and 18:3 were consumed specifically, reaching 50% after 24 h (5.5 mmol/g FW). Despite the high reactivity of hydroperoxides, the steady-state level of PUFA hydroperoxides after 24 h was shown to represent still 10% of PUFA consump-tion. In addition, infiltration of 18:2 into leaves, 16 h after cryptogein treatment, increased the 9-HODE level, indicating that 9-LOX activity was not substrate-saturated in vivo. Over-all, these results strongly suggest that massive PUFA con-sumption occurs mainly via the LOX pathway. Among the lipid changes following tobacco elicitation, previous investigations described a late decrease in galactolipid levels (15). In the present model, the analysis of the fatty acid composition of the lipid classes showed further that PUFAs originated mainly from galactolipids. Thus, chloroplasts appear as being the main source of PUFAs in the process. The key role of chloroplast lipids in synthesis of products derived from fatty acid hydroper-oxides, the so-called oxylipin pathway, was first established using mutants of PUFA synthesis for freezing-induced volatile aldehyde production (54). Furthermore, the metabolism site of fatty acid hydroperoxides was shown to be located on the en-velope membranes of chloroplasts (55), and it appeared that some pathogen- or MeJA-induced LOX genes are chloroplast-targeted (49, 56). In addition, morphological changes on chlo-roplasts were previously observed in various models of plant-pathogen interaction (6 – 8) and in cryptogein-treated tobacco leaves (57). The present results highlighted the major role of

this organelle in the development of leaf HR.

A tobacco 9-LOX was previously purified and characterized on cultured cells treated by an elicitor from the pathogen

Phy-tophthora parasitica var. nicotianae (51). The 9/13-LOX

speci-ficity (87/13 and 93/7 on 18:2 and 18:3 substrates, respectively) is consistent with our results on cryptogein-induced (9S)-LOX in leaves (98/2 on 18:2). The expression of the gene was shown to occur in elicited cell cultures and, furthermore, in plants upon infection (46). In the present work, a cryptogein-mediated expression of the same gene was demonstrated. From the chiral analyses, a low constitutive (13S)-LOX metabolism can be pro-posed in control leaves. Upon cryptogein treatment, the metab-olism is shifted toward a massive production of (9S)-fatty acid hydroperoxides. The initial low constitutive (13S)-LOX activity could be sufficient for the synthesis of JA, an early plant defense signaling compound considered to operate in plant pathogen interactions (25, 30, 31). The enzymes required for JA synthesis have been proposed as being expressed constitutively (58), and the 18:3 precursor might be provided by the induction of phospholipases, known to occur rapidly after elicitation (19, 59). JA was mentioned as being produced in elicited tobacco cells before 9-LOX activity induction and shown to mediate 9-LOX gene expression (46). In the present work, cryptogein-and MeJA-induced LOX activities were shown to exhibit the same 9S specificity. Thus, participation of JA in the signaling cascade leading to cryptogein-induced 9-LOX gene expression appears likely. In addition, early accumulation of JA was de-scribed in infected tobacco leaves undergoing HR (60). These results, taken together, suggest that at least two different LOXs, with 13 and 9 specificity, operating in signaling and in production of cell death effectors, respectively, should be in-volved in tobacco leaf HR.

During the development of cryptogein-elicited leaf necrosis, free fatty acid hydroperoxides were produced from membrane lipids, indicating the involvement of hydrolase activity. Indeed, a late induction of acyl hydrolase or lipase activities was ob-served in various models of plant pathogen interactions (26, 61). In general, LOX substrates are free fatty acids. However, in the early stages of seed germination, lipid body-associated LOX led to lipid peroxidation prior to hydrolase action (40). In addition, in elicited soybean seedlings, the induced LOX was shown to act in vitro on phospholipids (27). Thus, in the cryp-togein-induced tobacco HR, the question whether LOX acts before or after the hydrolase action was addressed. First, all hydroxy fatty acid isomers were characterized in esterified lipids whereas, consistent with the 9-LOX activity determined

FIG. 7. LOX substrate and product infiltration on the development of tobacco leaf necrosis. Leaves from control plants were infiltrated between 2 secondary leaf veins with various concentrations of compounds in 0.5% Tween 80. A, infiltration of increased concentrations of 13-hydroperoxide of 18:2 (0, 1, 2, 5, and 10 mM). B, comparison of 5 mMsubstrate reactivity was carried out on the same leaf by infiltration of 18:2 free fatty acid (18:2), tert-butyl hydroperoxide (tert-BuOOH), 13-hydroperoxide of 18:2 (HPODE), and the corresponding 13-hydroxy 18:2 fatty acid (HODE). C, leaves from 5 days MeJA-treated tobacco plants, as described under “Experimental Procedures”, infiltrated with 1, 2, 5, or 10 mM18:2 fatty acid. The symptoms presented in A–C were those observed after 4 h of incubation. Typical photographs from triplicate experiments leading to the same results were shown.

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in vitro on free fatty acids, only 9-positional isomers were

analyzed as free fatty acid hydroperoxides, suggesting that hydrolysis occurs before peroxidation. Second, free 16:3 fatty acid was shown can be peroxidized by elicited 9-LOX in vitro but 16:3 not was consumed and apparently not peroxidized in elicited leaves. Since 16:3 is exclusively located on the sn-2 position of chloroplast lipids (62), this last result could be explained assuming lipase(s) specific of the sn-1 position act before 9-LOX. Finally, the observation that MeJA-induced 9-LOX activity did not lead to either metabolite accumulation or to HR symptoms, in the absence of PUFA infiltration, also argues in favor of that hypothesis. This last result further suggests that lipase(s) might be induced by a signaling path-way different from JA. The characterization of elicited lipases, their product specificity, and the signaling pathway leading to their induction are currently under investigation.

The events leading to cryptogein-induced HR are tentatively summarized in Fig. 8. The induction of LOX pathway is an active process of membrane degradation leading to hypersen-sitive cell death. Lipase activity appears to be involved up-stream from 9-LOX action. JA was proposed as an early signal for 9-LOX gene induction. In the light of our results, further peroxidation of membrane lipids was considered as a conse-quence of massive production of fatty acid hydroperoxides, rather than AOS production during the early oxidative burst. The induction of the LOX peroxidative pathway was not de-scribed in mammalian PCD (63) and can be considered as a characteristic of plant HR-PCD. Apoptosis in mammalian cells and lipid peroxidation were correlated, both induced by oxida-tive agents, and both inhibited in transfected cells overexpress-ing the oncogene bcl-2 (64). Apoptosis was shown to be induced by free fatty acid hydroperoxides but was not repressed by the Bcl-2 protein, suggesting that the protein acts before lipid peroxidation (65). In plants, HR induction appears not depend-ent on the Bcl-2 family (12), and lipid peroxidation was shown in this work as being an active process.

In many aspects, the production of free fatty acid hydroper-oxides must be considered as an important part of the plant

defense response to limit pathogen invasion. Products from the LOX pathway include hydroxy fatty acids, observed in this work, which are substrates for cutin biosynthesis, reinforcing tissue defenses (66). Lipid hydroperoxides and derived prod-ucts are toxic to microorganisms (25, 30, 31, 50, 67). Finally, fatty acid hydroperoxides were also proposed as acting as sig-naling compounds to neighboring cells (25) and eliciting phy-toalexin synthesis (48, 66). A recent investigation (29), showing that tobacco plants exhibiting HR and a resistance toward the pathogen P. parasitica displayed susceptibility when antisense 9-LOX plants were infected, is in favor of that assumption.

The massive production of free fatty acid hydroperoxides observed in this work was shown to induce plant cell death and can also be considered as a defense mechanism against the invading pathogen. This metabolism has been observed during the development of leaf HR, and chloroplast lipids appear es-sential for the response. Among the questions arising from these results, further investigations must be focused to address the occurrence of this pathway in other plant-pathogen inter-actions, other tissues (i.e. non-photosynthetic tissues), and in the cultured cell model. The active process of membrane deg-radation described in this work, together with the induction of nucleases (5– 8) and proteases (9 –11), can be considered as one of the important features leading to the hypersensitive pro-grammed cell death.

Acknowledgments—We thank Dr. Michel Ponchet (from INRA,

France) for providing cryptogein and helpful discussions. We are also grateful to Michel Pe´an and co-workers for greenhouse facilities and to Christiane Richaud for help in preparing the manuscript.

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