Analyse du rôle du H2O2 et du stress oxydant intracellulaire dans les réponses aux agents pathogènes par l'usage d'un mutant conditionnel déficient en catalase et par profilage métabolique

Les formes actives de l'oxygène (FAO) constituent un élément clé pour la réponse des plantes aux stimuli de l'environnement parmi lesquels l'attaque par des agents pathogènes pour laquelle le rôle des FAO est bien étudié. Cependant, l'origine subcellulaire de production des FAO ainsi que l'impact des FAO produites dans différents compartiments subcellulaires au cours de la réponse aux agents pathogènes restent à élucider. Il en est de même concernant l'existence d'éventuelles interactions entre FAO d'origines subcellulaires différentes. Les peroxisomes constituent une source majeure de H2O2 produit au cours du processus de photorespiration et qui, chez Arabidopsis, est principalement métabolisé par la CATALASE2 (CAT2). En accord avec ceci, le mutant cat2 montre un phénotype conditionnel faisant apparaître des lésions spontanées sur ses feuilles lorsque cultivé en jours longs (JL) mais pas en jours courts (JC). L'un des buts de la présente étude était donc d'identifier des facteurs expliquant cette dépendance à la photopériode. Premièrement, il a été montré que la formation des lésions chez cat2 est associée avec une accumulation de glutathion oxydé et accompagnée par une activation de réponses de défense manifestées par une résistance accrue à une bactérie virulente. Malgré le stress oxydant induit par l'importante production de H2O2, les réponses de défense induites chez cat2, y compris la mort cellulaire, se sont révélées totalement dépendantes de l'acide salicylique (AS) synthétisé via la voie de l'isochorismate. Les réponses de défense absentes chez cat2 en JC ont été induites par la complémentation par l'AS. Deuxièmement, le profilage métabolique non ciblé m'a permis d'identifier des composés contrôlés par l'AS et/ou le H2O2. Particulièrement, les teneurs en myo-inositol (MI) étaient diminuées chez cat2 indépendamment de l'AS. La complémentation de cat2 cultivé en JL par du MI a supprimé la mort cellulaire et les réponses de défenses associées. L'inversion de ces réponses, par le blocage de la synthèse de l'AS ou par l'ajout de MI, n'a pas coincidée avec une diminution du stress oxydant mesuré par des transcrits marqueurs ou par la perturbation du glutathion. Troisièmement, le blocage, par une approche génétique, de l'accumulation du GSSG chez le double mutant cat2 cad2 indique un rôle joué par le glutathion dans les réponses dépendantes de l'AS. Ceci a été souligné par l'étude du mutant gr1 affecté pour une glutathion réductase. Finalement, afin d'étudier l'existence d'interactions entre les FAO intracellulaires et la fonction des NADPH oxydases, les double mutants cat2 atrbohD et cat2 atrbohF ont été produits. Il s'est révélé que les deux NADPH oxydases interagissaient avec les FAO intracellulaires pour spécifiquement contrôler des réponses dépendantes de l'AS, incluant la mort cellulaire. Ainsi, en se basant sur nos données et les connaissances acquises sur les interactions biotiques, nous avons émis une hypothèse décrivant un potentiel dialogue entre les FAO intra et extraclullaires survenant au cours des interactions plantes-pathogènes impliquant l'AS.Reactive oxygen species (ROS) are key players in environmental signalling, and their roles in pathogen responses are weil described. However, the subcellular origin of ROS production during pathogen responses, as weil as the impact of ROS produced at different sites, remain to be established. The peroxisomes are a major source of intracellular HzOz generated in processes such as photorespiration, and previous studies have shown that, in Arabidopsis, this HzOz is metabolized primarily by CAT ALASE2 (CAT2). Accordingly, cat2 knockouts show a conditional phenotype in which lesions appear on the leaves of the mutant grown in long days (LD) but not short days (SD). The present study investigated the factors underlying this daylength-dependent response. First, it is shown that lesion formation in cat2 is associated with accumulation of oxidized glutathione (GSSG), and accompanied by activation of defence responses and enhanced resistance to virulent bacteria. Despite the oxidative stress triggered by increased intracellular H20Z production in cat2, defence responses, including cell death, are totally dependent on salicylic acid (SA) synthesis through the isochorismate pathway. The failure of cat2 to activate defence responses in SD can be complemented by exogenous SA. Second, non-targeted metabolite profiling identified cbmpounds under the control of SA and/or HzOz. ln particular, myo-inositol (MI) was identified as a compound decreased in cat2 in an SA-independent manner. Complementation of cat2 growing in LD with MI abrogated cell death and associated defence responses. Reversion of these responses by blocking SA synthesis or by supplying MI was not associated with decreased oxidative stress measured as marker transcript accumulation or glutathione perturbation. Third, genetically blocking accumulation of GSSG in cat2 cad2 double mutants pointed to a role for glutathionein SA-dependent responses, and this was underscored by analysis of gr] knockouts for glutathione reductase. Finally, to investigate possible interactions between intracellular ROS production and NADPH oxidase function, double cat2 atrboh mutants were produced and biotic stress responses and metabolite profiles compared to the parent single mutants. Both NADPH oxidases were shown to interact with intracellular ROS to specifically control SA-dependent responses, including cell death. Based on these and literature data, a hypothesis is constructed to describe possible crosstalk between extracellular and intracellular ROS during SA-dependent plant-pathogen interactions.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Catalase function in plants: a focus on Arabidopsis mutants as stress-mimic models

Hydrogen peroxide (H2O2) is an important signal molecule involved in plant development and environmental responses. Changes in H2O2 availability can result from increased production or decreased metabolism. While plants contain several types of H2O2-metabolizing proteins, catalases are highly active enzymes that do not require cellular reductants as they primarily catalyse a dismutase reaction. This review provides an update on plant catalase genes, function, and subcellular localization, with a focus on recent information generated from studies on Arabidopsis. Original data are presented on Arabidopsis catalase single and double mutants, and the use of some of these lines as model systems to investigate the outcome of increases in intracellular H2O2 are discussed. Particular attention is paid to interactions with cell thiol-disulphide status; the use of catalase-deficient plants to probe the apparent redundancy of reductive H2O2-metabolizing pathways; the importance of irradiance and growth daylength in determining the outcomes of catalase deficiency; and the induction of pathogenesis-related responses in catalase-deficient lines. Within the context of strategies aimed at understanding and engineering plant stress responses, the review also considers whether changes in catalase activities in wild-type plants are likely to be a significant part of plant responses to changes in environmental conditions or biotic challenge

Peroxisomal Hydrogen Peroxide Is Coupled to Biotic Defense Responses by ISOCHORISMATE SYNTHASE1 in a Daylength-Related Manner1[C][W][OA]

While it is well established that reactive oxygen species can induce cell death, intracellularly generated oxidative stress does not induce lesions in the Arabidopsis (Arabidopsis thaliana) photorespiratory mutant cat2 when plants are grown in short days (SD). One interpretation of this observation is that a function necessary to couple peroxisomal hydrogen peroxide (H2O2)-triggered oxidative stress to cell death is only operative in long days (LD). Like lesion formation, pathogenesis-related genes and camalexin were only induced in cat2 in LD, despite less severe intracellular redox perturbation compared with SD. Lesion formation triggered by peroxisomal H2O2 was modified by introducing secondary mutations into the cat2 background and was completely absent in cat2 sid2 double mutants, in which ISOCHORISMATE SYNTHASE1 (ICS1) activity is defective. In addition to H2O2-induced salicylic acid (SA) accumulation, the sid2 mutation in ICS1 abolished a range of LD-dependent pathogen responses in cat2, while supplementation of cat2 with SA in SD activated these responses. Nontargeted transcript and metabolite profiling identified clusters of genes and small molecules associated with the daylength-dependent ICS1-mediated relay of H2O2 signaling. The effect of oxidative stress in cat2 on resistance to biotic challenge was dependent on both growth daylength and ICS1. We conclude that (1) lesions induced by intracellular oxidative stress originating in the peroxisomes can be genetically reverted; (2) the isochorismate pathway of SA synthesis couples intracellular oxidative stress to cell death and associated disease resistance responses; and (3) camalexin accumulation was strictly dependent on the simultaneous presence of both H2O2 and SA signals

Differential gene expression and metabolomic analyses of Brachypodium distachyon infected by deoxynivalenol producing and non-producing strains of Fusarium graminearum

Background: Fusarium Head Blight (FHB) caused primarily by Fusarium graminearum (Fg) is one of the major diseases of small-grain cereals including bread wheat. This disease both reduces yields and causes quality losses due to the production of deoxynivalenol (DON), the major type B trichothecene mycotoxin. DON has been described as a virulence factor enabling efficient colonization of spikes by the fungus in wheat, but its precise role during the infection process is still elusive. Brachypodium distachyon (Bd) is a model cereal species which has been shown to be susceptible to FHB. Here, a functional genomics approach was performed in order to characterize the responses of Bd to Fg infection using a global transcriptional and metabolomic profiling of B. distachyon plants infected by two strains of F. graminearum: a wild-type strain producing DON (Fg DON+) and a mutant strain impaired in the production of the mycotoxin (Fg DON-). Results: Histological analysis of the interaction of the Bd21 ecotype with both Fg strains showed extensive fungal tissue colonization with the Fg DON+ strain while the florets infected with the Fg DON- strain exhibited a reduced hyphal extension and cell death on palea and lemma tissues. Fungal biomass was reduced in spikes inoculated with the Fg DON- strain as compared with the wild-type strain. The transcriptional analysis showed that jasmonate and ethylene-signalling pathways are induced upon infection, together with genes encoding putative detoxification and transport proteins, antioxidant functions as well as secondary metabolite pathways. In particular, our metabolite profiling analysis showed that tryptophan-derived metabolites, tryptamine, serotonin, coumaroyl-serotonin and feruloyl-serotonin, are more induced upon infection by the Fg DON+ strain than by the Fg DON- strain. Serotonin was shown to exhibit a slight direct antimicrobial effect against Fg. Conclusion: Our results show that Bd exhibits defense hallmarks similar to those already identified in cereal crops. While the fungus uses DON as a virulence factor, the host plant preferentially induces detoxification and the phenylpropanoid and phenolamide pathways as resistance mechanisms. Together with its amenability in laboratory conditions, this makes Bd a very good model to study cereal resistance mechanisms towards the major disease FHB

Glutathione

Glutathione is a simple sulfur compound composed of three amino acids and the major non-protein thiol in many organisms, including plants. The functions of glutathione are manifold but notably include redox-homeostatic buffering. Glutathione status is modulated by oxidants as well as by nutritional and other factors, and can influence protein structure and activity through changes in thiol-disulfide balance. For these reasons, glutathione is a transducer that integrates environmental information into the cellular network. While the mechanistic details of this function remain to be fully elucidated, accumulating evidence points to important roles for glutathione and glutathione-dependent proteins in phytohormone signaling and in defense against biotic stress. Work in Arabidopsis is beginning to identify the processes that govern glutathione status and that link it to signaling pathways. As well as providing an overview of the components that regulate glutathione homeostasis (synthesis, degradation, transport, and redox turnover), the present discussion considers the roles of this metabolite in physiological processes such as light signaling, cell death, and defense against microbial pathogen and herbivores