Singlet oxygen triggers chloroplast rupture and cell death in the zeaxanthin epoxidase defective mutant aba1 of Arabidopsis thaliana under high light stress

[EN] The two Arabidopsis thaliana mutants, aba1 and max4, were previously identified as sharing a number of coregulated genes with both the flu mutant and Arabidopsis cell suspension cultures exposed to high light (HL). On this basis, we investigated whether aba1 and max4 were generating high amounts of singlet oxygen (1O2) and activating 1O2-mediated cell death. Thylakoids of aba1 produced twice as much 1O2 as thylakoids of max4 and wild type (WT) plants when illuminated with strong red light. 1O2 was measured using the spin probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride. 77-K chlorophyll fluorescence emission spectra of thylakoids revealed lower aggregation of the light harvesting complex II in aba1. This was rationalized as a loss of connectivity between photosystem II (PSII) units and as the main cause for the high yield of 1O2 generation in aba1. Upregulation of the 1O2 responsive gene AAA-ATPase was only observed with statistical significant in aba1 under HL. Two early jasmonate (JA)-responsive genes, JAZ1 and JAZ5, encoding for two repressor proteins involved in the negative feedback regulation of JA signalling, were not up-regulated to the WT plant levels. Chloroplast aggregation followed by chloroplast rupture and eventual cell death was observed by confocal imaging of the fluorescence emission of leaf cells of transgenic aba1 plants expressing the chimeric fusion protein SSU-GFP. Cell death was not associated with direct 1O2 cytotoxicity in aba1, but rather with a delayed stress response. In contrast, max4 did not show evidence of 1O2-mediated cell death. In conclusion, aba1 may serve as an alternative model to other 1O2-overproducing mutants of Arabidopsis for investigating 1O2-mediated cell death

Photosynthetic electron flow affects H2O2 signaling by inactivation of catalase in Chlamydomonas reinhardtii

A specific signaling role for H2O2 in Chlamydomonas reinhardtii was demonstrated by the definition of a promoter that specifically responded to this ROS. Expression of a nuclear-encoded reporter gene driven by this promoter was shown to depend not only on the level of exogenously added H2O2 but also on light. In the dark, the induction of the reporter gene by H2O2 was much lower than in the light. This lower induction was correlated with an accelerated disappearance of H2O2 from the culture medium in the dark. Due to a light-induced reduction in catalase activity, H2O2 levels in the light remained higher. Photosynthetic electron transport mediated the light-controlled down-regulation of the catalase activity since it was prevented by 3-(3′4′-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. In the presence of light and DCMU, expression of the reporter gene was low while the addition of aminotriazole, a catalase inhibitor, led to a higher induction of the reporter gene by H2O2 in the dark. The role of photosynthetic electron transport and thioredoxin in this regulation was investigated by using mutants deficient in photosynthetic electron flow and by studying the correlation between NADP-malate dehydrogenase and catalase activities. It is proposed that, contrary to expectations, a controlled down-regulation of catalase activity occurs upon a shift of cells from dark to light. This down-regulation apparently is necessary to maintain a certain level of H2O2 required to activate H2O2-dependent signaling pathways

Singlet oxygen production in photosynthesis

A photosynthetic organism is subjected to photooxidative stress when more light energy is absorbed than is used in photosynthesis. In the light, highly reactive singlet oxygen can be produced via triplet chlorophyll formation in the reaction centre of photosystem II and in the antenna system. In the antenna, triplet chlorophyll is produced directly by excited singlet chlorophyll, while in the reaction centre it is formed via charge recombination of the light-induced charge pair. Changes of the mid-point potential of the primary quinone acceptor in photosystem II modulate the pathway of charge recombination in photosystem II and influence the yield of singlet oxygen production. Singlet oxygen can be quenched by b-carotene, a-tocopherol or can react with the D1 protein of photosystem II as target. If not completely quenched, it can specifically trigger the up-regulation of the expression of genes which are involved in the molecular defence response of plants against photo-oxidative stress. Key words: Light energy, photo-oxidative stress, photosynthesis, photosystem II, singlet oxygen

Enzymes impliqués dans la production des formes réactives de l'oxygène dans les membranes plasmiques, les mitochondries et les chloroplastes

Les formes réactives de l oxygène (FRO) ont été analysées dans différents compartiments cellulaires en utilisant des méthodes spectroscopiques (UV/VIS, fluorescence, infrarouge, résonance paramagnétique électronique). L identité et les mécanismes catalytiques des enzymes qui produisent les FRO dans les membranes plasmiques (MP) et les mitochondries ont été étudiés, ainsi que le rôle protectif de l oxydase terminale plastidiale (PTOX) des chloroplastes. Cd2+ s est révélé être un inhibiteur de la NADPH oxydase des MP. In vivo Cd2+ inhibait la production extracellulaire de O2.- mais stimulait l accumulation de H2O2. Dans des mitochondries isolées, Cd2+ a augmenté la production de FRO . Antimycin A a entrainé une élévation du H2O2 extracellulaire, confirmant que la mitochondrie est le site principal de production de l H2O2 extracellulaire induite par Cd2+ in vivo. Une quinone réductase (QR) génératrice de FRO a été isolée des MP. La déprotonation PH dépendante du quinole a produit des formes intermédiaires instables qui génèrent des FRO par réaction avec O2. Des espèces quinoniques ont été détectées dans la MP et pourraient servir de substrat aux QR in vivo. La protection de la chaine photosynthétique de transfert d électron par la plastoquinol ; O2 oxydoréductase a été étudiée chez des plantes PTOX+ surexprimant PTOX. En raison de leur réponse altérée en conditions de faible et de forte intensité lumineuse, il a été proposé que pour fonctionner comme enzyme protectrice, PTOX est couplée à une SOD. Chez les lignées PTOX+, le niveau de SOD chloroplastique n était pas plus élevé, limitant probablement leur capacité à détoxifier les taux élevés de O2.- généré.Production of reactive oxygen species (ROS) was studied in different subcellular compartments using spectroscopic methods (UV/VIS, fluorescence, infrared and electron paramagnetic resonance). The identities and catalytic mechanisms of ROS-producing enzymes in the plasma membrane (PM) were studied as well as mitochondrial ROS accumulation in response to cadmium (Cd2+) and the protectrice role of the plastid terminal oxidase (PTOX). Cd2+ was shown to be an inhibitor of the PM superoxide (O2.-) producing NADPH oxidase in vitro. In vivo Cd2+ inhibited the extracellular production of O2.- but stimulated the accumulation of hydrogen peroxide (H2O2 ). Cd2+ induced ROS production in isolated mitochondria. Mitochondrial ROS- inducing inhibitors increased extracellular H2O2 production confirming these organelles the main source of Cd2+ induced extracellular H2O2 generation in vivo. A ROS-producing quinone reductase(QR) was isolated from PMs. ROS production occurred via the pH dependent deprotonation of the end product menadiol leading to its intermediate forms that react with O2 forming O2.- and H2O2. A potential naphthoquinose species in PMs was identified that could serve as a QR substrate in vivo. The protection of the photosynthetic electron transfer chain by the PTOX, a plastoquinol : O2 oxidoreductase, was studied in PTOX-overexpressing plants (PTOX+ ). Based on the altered response to low and high light conditions in PTOX+ it was proposed that PTOX is coupled to an SOD in order to function as a protective enzyme. The absence of additional chloroplastic SOD in PTOX+ lines might have limited the plant s ability to detoxify O2.- produced by elevated levels of PTOX+ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Importing Manganese into the Chloroplast: Many Membranes to Cross

International audienc

Regulation of the generation of reactive oxygen species during photosynthetic electron transport

International audienceLight capture by chlorophylls and photosynthetic electron transport bury the risk of the generation of reactive oxygen species (ROS) including singlet oxygen, superoxide anion radicals and hydrogen peroxide. Rapid changes in light intensity, electron fluxes and accumulation of strong oxidants and reductants increase ROS production. Superoxide is mainly generated at the level of photosystem I while photosystem II is the main source of singlet oxygen. ROS can induce oxidative damage of the photosynthetic apparatus, however, ROS are also important to tune processes inside the chloroplast and participate in retrograde signalling regulating the expression of genes involved in acclimation responses. Under most physiological conditions light harvesting and photosynthetic electron transport are regulated to keep the level of ROS at a non-destructive level. Photosystem II is most prone to photoinhibition but can be quickly repaired while photosystem I is protected in most cases. The size of the transmembrane proton gradient is central for the onset of mechanisms that protect against photoinhibition. The proton gradient allows dissipation of excess energy as heat in the antenna systems and it regulates electron transport. pH-dependent slowing down of electron donation to photosystem I protects it against ROS generation and damage. Cyclic electron transfer and photoreduction of oxygen contribute to the size of the proton gradient. The yield of singlet oxygen production in photosystem II is regulated by changes in the midpoint potential of its primary quinone acceptor. In addition, numerous antioxidants inside the photosystems, the antenna and the thylakoid membrane quench or scavenge ROS

The Dual Role of the Plastid Terminal Oxidase PTOX: Between a Protective and a Pro-oxidant Function.

International audienc

Reactive oxygen intermediates produced by photosynthetic electron transport are enhanced in short-day grown plants

AbstractLeaves of tobacco plants grown in short days (8h light) generate more reactive oxygen species in the light than leaves of plants grown in long days (16h light). A two fold higher level of superoxide production was observed even in isolated thylakoids from short day plants. By using specific inhibitors of photosystem II and of the cytochrome b6f complex, the site of O2 reduction could be assigned to photosystem I. The higher rate of O2 reduction led to the formation of a higher proton gradient in thylakoids from short day plants. In the presence of an uncoupler, the differences in O2 reduction between thylakoids from short day and long day plants were abolished. The pigment content and the protein content of the major protein complexes of the photosynthetic electron transport chain were unaffected by the growth condition. Addition of NADPH, but not of NADH, to coupled thylakoids from long day plants raised the level of superoxide production to the same level as observed in thylakoids from short day plants. The hypothesis is put forward that the binding of an unknown protein permits the higher rate of pseudocyclic electron flow in thylakoids from short-day grown plants and that this putative protein plays an important role in changing the proportions of linear, cyclic and pseudocyclic electron transport in favour of pseudocyclic electron transport. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Articifical