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.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 nonpathogen. 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 (1, 2). 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 (3, 4). 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 (1, 2). 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 calciumdependent endonucleases (5-8) and of cysteine proteases (9 -11), and involvement of similar regulation factors (2). Some differences between HR and mammalian apoptosis were observed, however, such as changes in DNA laddering (5,8) and the lack in HR of the repressor role of Bcl-x L (12). One ultimate characteristic of HR is the loss of membrane integrity, and thus HR is often characterized by an associated electrolyte le...

We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species (ROS)-mediated lipid peroxidation from 9-and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H 2 O 2 -dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H 2 O 2 plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H 2 O 2 and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H 2 O 2 and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.Different defense mechanisms are used by plants to cope with pathogen assaults. A major source of resistance is conditioned by the interaction between plant resistance and pathogen avirulence gene products (Martin et al., 2003;Rathjen and Moffett, 2003). This defense strategy is characterized by (1) the activation of the hypersensitive response (HR), typified by a localized programmed cell death activation; (2) the initiation of defense responses, including cell wall reinforcement, accumulation of phytoalexins, and expression of antimicrobial proteins; and (3) the onset of a local and systemic acquired resistance (Lamb and Dixon, 1997;Beers and McDowell, 2001;Greenberg and Yao, 2004).The signaling cascades leading to the HR are starting to be elucidated. Several resistance genes have been cloned, and protein kinases, phosphatases, and GTP-binding proteins have all been implicated downstream of these recognition proteins (Martin et al., 2003;Rathjen and Moffett, 2003). Changes in ion fluxes across the plasma membrane and the production of reactive oxygen species (ROS) and nitric oxide (NO) are among the earliest events following pathogen infection or elicitor treatment in cultured plant cells (Doke, 1997;Grant and Loake, 2000;Wendehenne et al., 2004). The production of ROS is biphasic, with a sustained second oxidative burst in response to an avirulent pathogen, and monophasic and transient with a virulent pathogen. This suggests that ROS production may be responsible for some of the defense-associated processes (Lamb and Dixon, 1997). Furthermore, ROS, and specifically H 2 O 2 , are key modulators of NO in triggering plant cell death (Delledonne et al., 2001;Neill et al., 2002;Wendehenne et al., 2004).In order to obtain further insight into the role of H 2 O 2 in plant signaling and cell death, we used transgenic tobacco ...

Cadmium is suspected to exert its toxic action on cells through oxidative damage. However, the transition metal is unable to directly generate reactive oxygen species (ROS) via redox reactions with molecular oxygen in a biological environment. Here, we show that bright yellow-2 (BY-2) tobacco cells exposed to millimolar concentrations of CdCl 2 developed cell death within 2-3 h. The death process was preceded by two successive waves of ROS differing in their nature and subcellular localization. Firstly, these consisted in the transient NADPH oxidase-dependent accumulation of H 2 O 2 followed by the accumulation of O 2 -. in mitochondria. A third wave of ROS consisting in fatty acid hydroperoxide accumulation was concomitant with cell death. Accumulation of H 2 O 2 was preceded by an increase in cytosolic free calcium concentration originating from internal pools that was essential to activate the NADPH oxidase. The cell line gp3, impaired in NADPH oxidase activity, and that was unable to accumulate H 2 O 2 in response to Cd 2 + , was nevertheless poisoned by the metal. Therefore, this first wave of ROS was not sufficient to trigger all the cadmiumdependent deleterious effects. However, we show that the accumulation of O 2 -. of mitochondrial origin and membrane peroxidation are key players in Cd 2 + -induced cell death.

). † These authors contributed equally to this work. SummaryVarious physiological imbalances lead to reactive oxygen species (ROS) overproduction and/or increases in lipoxygenase (LOX) activities, both events ending in lipid peroxidation of polyunsaturated fatty acids (PUFAs). Besides the quantification of such a process, the development of tools is necessary in order to allow the identification of the primary cause of its development and localization. A biochemical method assessing 9 LOX, 13 LOX and ROS-mediated peroxidation of membrane-bound and free PUFAs has been improved. The assay is based on the analysis of hydroxy fatty acids derived from PUFA hydroperoxides by both the straight and chiral phase high-performance liquid chromatography. Besides the upstream products of peroxidation of the 18:2 and 18:3 PUFAs, products coming from the 16:3 were characterized and their steady-state level quantified. Moreover, the observation that the relative amounts of the ROS-mediated peroxidation isomers of 18:3 were constant in leaves allowed us to circumvent the chiral analyses for the discrimination and quantification of 9 LOX, 13 LOX and ROS-mediated processes in routine experiments. The methodology has been successfully applied to decipher lipid peroxidation in Arabidopsis leaves submitted to biotic and abiotic stresses. We provide evidence of the relative timing of enzymatic and non-enzymatic lipid peroxidation processes. The 13 LOX pathway is activated early whatever the nature of the stress, leading to the peroxidation of chloroplast lipids. Under cadmium stress, the 9 LOX pathway added to the 13 LOX one. ROS-mediated peroxidation was mainly driven by light and always appeared as a late process.

The first two authors contributed equally to this work. SummaryLipid peroxidation, often associated with hypersensitive cell death, may be initiated either by active oxygen species (AOS) or lipoxygenases (LOX). Here we report a detailed analysis of this oxidative process in both incompatible and compatible interactions between the cotton cultivar Reba B50 and Xanthomonas campestris pv. malvacearum (Xcm). The hypersensitive reaction (HR) was characterized by a massive production of polyunsaturated fatty acid (PUFA) hydroperoxides together with typical tissue dehydration. Among these, isomers peroxidized on carbon 9, largely predominant, were chiral, showing an excess in the S enantiomer. The HR process was accompanied by an increase in 9S-LOX activity and preceded by transcription of a LOX gene (GhKLox1). These results showed that: (i) AOS produced during the oxidative burst were not involved in PUFA peroxidation during HR; and (ii) as previously described in elicited leaves of tobacco, the massive enzymatic lipid peroxidation was closely associated with hypersensitive cell death. During disease development in this cotton cultivar, the 9-lipoxygenation of PUFAs was late, weak, preceded by a faint accumulation of GhKLox1 transcripts, and associated with chlorosis but not with necrosis. Consequently, the main difference between incompatible and compatible interactions was in the precocity and intensity of the oxidative process, rather than in its nature. These data provide the evidence for a correlation between lipid peroxidation and hypersensitive cell death induced by pathogens.