Mitochondrial respiratory pathways modulate nitrate sensing and nitrogen-dependent regulation of plant architecture in Nicotiana sylvestris

Mitochondrial electron transport pathways exert effects on carbon–nitrogen (C/N) relationships. To examine whether mitochondria–N interactions also influence plant growth and development, we explored the responses of roots and shoots to external N supply in wild-type (WT) Nicotiana sylvestris and the cytoplasmic male sterile II (CMSII) mutant, which has a N-rich phenotype. Root architecture in N. sylvestris seedlings showed classic responses to nitrate and sucrose availability. In contrast, CMSII showed an altered ‘nitrate-sensing’ phenotype with decreased sensitivity to C and N metabolites. The WT growth phenotype was restored in CMSII seedling roots by high nitrate plus sugars and in shoots by gibberellic acid (GA). Genome-wide cDNA-amplified fragment length polymorphism (AFLP) analysis of leaves from mature plants revealed that only a small subset of transcripts was altered in CMSII. Tissue abscisic acid content was similar in CMSII and WT roots and shoots, and growth responses to zeatin were comparable. However, the abundance of key transcripts associated with GA synthesis was modified both by the availability of N and by the CMSII mutation. The CMSII mutant maintained a much higher shoot/root ratio at low N than WT, whereas no difference was observed at high N. Shoot/root ratios were strikingly correlated with root amines/nitrate ratios, values of <1 being characteristic of high N status. We propose a model in which the amine/nitrate ratio interacts with GA signalling and respiratory pathways to regulate the partitioning of biomass between shoots and roots

Implication de la mitochondrie végétale dans l'état redox cellulaire et la réponse au stress biotique

L'étude de l'apoptose, une forme de mort cellulaire programmée (PCD), a mis en évidence le rôle de la mitochondrie animale comme un site majeur de production de formes activées de l'oxygène (FAO) et un centre d'intégration des signaux cellulaires. La possibilité d'un rôle similaire pour la mitochondrie végétale n'a soulevé initialement que peu d'attention, étant donné la faible contribution intrinsèque des mitochondries végétales à la production de FAO. Dans ce travail, le rôle de la mitochondrie végétale dans la réponse au stress biotique a été étudié par une approche génétique originale, basée sur l'analyse du mutant mitochondrial CMSII de Nicotiana sylvestris. Le complexe I (NADH: ubiquinone oxydoréductase) de la chaîne respiratoire de ce mutant est non-fonctionnel suite à une délétion du gène mitochondrial nad7. Nous avons montré qu'à l'état constitutif, le dysfonctionnement mitochondrial entraîne, dans les cellules photosynthétiques, une modification de l'expression de défenses cellulaires (défenses antioxydantes, voie des phénylpropanoïdes). Une signalisation, issue directement ou indirectement de la mitochondrie, qui module l'expression de ces gènes nucléaires, est ainsi mise en évidence. Dans différents types de stress biotiques (réaction hypersensible induite par un éliciteur bactérien ou le VMT; maladie induite par une souche compatible du VMT ou Botrytis cinerea), la réponse du mutant diffère de celle de la plante WT. Nous proposons que le dysfonctionnement du complexe I confère à la plante un "état d'éveil" qui (1) modifierait son niveau seuil d'activation de la RH (2) lui permettrait de mettre en route plus rapidement certaines réponses de défense. Ce dysfonctionnement serait préjudiciable dans le cadre de la maladie, peut-être en relation avec des déficiences métaboliques. L'ensemble de ces observations met en évidence l'implication de la mitochondrie dans la perception et l'intégration des signaux lors d'un stress biotique chez une plante supérieure.The study of apoptosis, a kind of programmed cell death (PCD), pointed out the role of animal mitochondria as a major source of activated oxygen species (AOS) and an integrated centre of cellular signals. Such a role for plant mitochondria has been the subject of poor attention so far, due to their minor contribution in AOS generation in photosynthetic cells. In this work, we studied the role of mitochondria in biotic stress responses by an original genetic approach, based on the analysis of the Nicotiana sylvestris CMSII mitochondrial mutant. In this mutant, the respiratory complex I (NADH: ubiquinone oxidoreductase) is inactivated following a deletion in the mitochondrial genome of the nad7 gene. We showed that in non stressed plants, the mitochondrial dysfunction leads, in photosynthetic cells, to a modification of cellular defences (antioxidant defences, phenylpropanoid pathway). These results demonstrated the existence of a cellular signal originated directly or indirectly from mitochondrial respiratory chain, which modulated nuclear genes expression. Using different kinds of biotic stress (hypersensitive reaction induced by a bacterial elicitor or the TMV; disease induced by a compatible TMV strain and Botrytis cinerea), the CMS responses were shown to be different from those of WT plant. We propose that the complex I dysfunction confers an "alerted" state in the CMS plant, which (1) could modify its threshold of HR activation (2) could allow faster defence responses. This dysfunction may be detrimental in the case of disease, with a probable link to metabolic deficiencies. Altogether, these results point out the involvement of higher plant mitochondria in signal perception and integration during biotic stress.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Conséquences d'un défaut mitochondrial sur la réponse hypersensible (étude d'un mutant respiratoire de Nicotiana sylvestris)

La réponse hypersensible (HR) est considérée comme une forme de mort cellulaire programmée (PCD) chez les végétaux. Chez les animaux la mitochondrie joue un rôle important de signalisation et d'exécution dans la PCD. Afin d'évaluer l'implication de la mitochondrie au cours de la HR, le génotype sauvage (WT) et le mutant mitochondrial (CMSII) de Nicotiana sylvestris ont été examinés pour leur réponse à une protéine bactérienne élicitrice de la RH, la harpine N de Erwinia amylovora.Nous avons confirmé la présence de marqueurs de PCD animaux dans notre système: importance de l'influx de calcium, sortie précoce du cytochrome c hors des mitochondries et fragmentation des noyaux. L'ajout de calcium sur mitochondries in vitro entraîne la sortie du cytochrome c, qui pourrait s'effectuer suite à un changement de perméabilité des membranes mitochondriales. Nous avons mis en évidence une augmentation de la respiration totale in vivo durant la HR induite par la harpine. Chez le WT cette augmentation est principalement due à une forte augmentation de l'activité de la voie alterne de respiration (AOX) alors que chez CMSII les voies principale (COX) et AOX augmentent en parallèle. L'augmentation de la respiration pourrait entraîner une production accrue de Formes Activées de l'Oxygène intervenant dans la signalisation de la HR.Enfin, la harpine induit rapidement une chute de la photosynthèse et du rendement quantique du photosystème II ainsi qu'une accumulation de H2O2 dans les chloroplastes.Ces résultats mettent en évidence la participation des différents compartiments cellulaires à la PCD. La réponse du mutant CMSII à la harpine met en jeu des réponses différentes localisées au niveau de la mitochondrie ainsi qu'au niveau sub-cellulaire (davantage de transcrits antioxydants cytosoliques, fragmentation des noyaux moins importante) mais n'empêche pas in fine la HR.The hypersensitive response (HR) is considered to be a form of programmed cell death (PCD) in plants. In animals mitochondria play a major role in the signalling and execution of PCD. In order to evaluate the mitochondria involvement in HR, Nicotiana sylvestris WT genotype and mitochondrial mutant CMSII plants were studied for their reaction to the HR-inducing bacterial elicitor harpin from Erwinia amylovora.We confirm that some animal PCD markers occur in our system: importance of calcium influx, early cytochrome c exit from mitochondria intermembrane space and nuclei fragmentation. The addition of calcium onto mitochondria in vitro leads to the release of cytochrome c possibly mediated by changes in mitochondrial membranes permeability. We measured an in vivo increase in total respiration during the harpin-induced HR. In WT, this increase is mainly due to a strong increase in AOX activity whereas both AOX and cytochrome (COX) pathways increased in parallel in CMSII. The increase in respiration may drive a mitochondria-ROS overproduction involved in HR signalling. The harpin also induced an early decrease in photosynthesis and quantic yield of photosystem II and H2O2 accumulation in chloroplasts.These results highlight the involvement of different cellular compartments in PCD. The CMSII reaction to harpin involves different responses localized to the mitochondrial level and sub cellular levels (more cytosolic antioxidative transcripts, less nuclei fragmentation) but cannot in fine counteract the HR.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Mitochondrial complex I dysfunction increases CO₂ efflux and reconfigures metabolic fluxes of day respiration in tobacco leaves

Mutants affected in complex I are useful to understand the role played by mitochondrial electron transport and redox metabolism in cellular homeostasis and signaling. However, their respiratory phenotype is incompletely described and a specific examination of day respiration (Rd) is lacking. Here, we used isotopic methods and metabolomics to investigate the impact of complex I dysfunction on Rd in two respiratory mutants of forest tobacco (Nicotiana sylvestris): cytoplasmic male sterile II (CMSII) and nuclear male sterile 1 (NMS1), previously characterized for complex I disruption. Rd was higher in mutants and the inhibition of leaf respiration by light was lower. Higher Rd values were caused by increased (phosphoenol)pyruvate (PEP) metabolism at the expense of anaplerotic (PEP carboxylase (PEPc) ‐catalyzed) activity. De novo synthesis of Krebs cycle intermediates in the light was larger in mutants than in the wild‐type, although numerically small in all genotypes. Carbon metabolism in mutants involved alternative pathways, such as alanine synthesis, and an increase in amino acid production with the notable exception of aspartate. Our results show that the alteration of NADH re‐oxidation activity by complex I does not cause a general inhibition of catabolism, but rather a re‐orchestration of fluxes in day respiratory metabolism, leading to an increased CO2 efflux

Interactions mitochondries-chloroplastes: Apports de l' étude d’un mutant mitochondrial de Nicotiana sylvestris.

International audienc

Interactions mitochondries-chloroplastes: Apports de l' étude d’un mutant mitochondrial de Nicotiana sylvestris.

International audienc

Répercussions d'un défaut mitochondrial sur les interactions subcellulaires dans le métabolisme foliaire (analyse d'un mutant respiratoire de Nicotiana Sylvestris)

LE DIALOGUE EXISTANT ENTRE LES DIFFERENTS COMPARTIMENTS SUBCELLULAIRES EST BASE SUR DES ECHANGES MASSIFS DE METABOLITES, D'ATP ET DE POUVOIR REDUCTEUR. LA CHAINE DE TRANSPORT D'ELECTRONS MITOCHONDRIALE JOUE UN ROLE IMPORTANT DANS CETTE COORDINATION INTER-COMPARTIMENTALE. CE TRAVAIL PRESENTE UNE ETUDE DE L'IMPLICATION DU PROCESSUS MITOCHONDRIAL DANS LES GRANDES FONCTIONS FOLIAIRES. LE MUTANT DE NICOTIANA SYLVESTRIS, CMSII (CYTOPLASMIC MALE-STERILE II), EST L'UN DES RARES MUTANTS DISPONIBLES AFFECTES DANS LE FONCTIONNEMENT D'UN COMPLEXE DE LA CHAINE RESPIRATOIRE (PERTE DU COMPLEXE I) SUITE A LA DELETION DU GENE MITOCHONDRIAL NAD7. CE MUTANT EST DONC UN EXCELLENT SYSTEME POUR DETERMINER LES CONSEQUENCES DE LA PERTE D'UNE DESHYDROGENASE MITOCHONDRIALE MAJEURE, AU NIVEAU DES PROCESSUS FOLIAIRES. IL EST MONTRE QUE LE DYSFONCTIONNEMENT DU COMPLEXE I INDUIT UN AJUSTEMENT DES SYSTEMES ANTIOXYDANTS, METTANT EN EVIDENCE UNE SIGNALISATION MITOCHONDRIE-NOYAU IMPLIQUEE DANS LA DETERMINATION DE LA RESISTANCE AU STRESS. DE PLUS, LE COMPLEXE I EST IMPORTANT DANS L'OPTIMISATION DE L'ASSIMILATION DU CO2 LORSQUE LA PHOTORESPIRATION EST ACTIVE, ET PARTICULIEREMENT PENDANT LA PERIODE D'INDUCTION QUI SUIT UNE TRANSITION OBSCURITE-LUMIERE. NOS DONNEES SUGGERENT QUE CE ROLE EST LIE A UNE OPTIMISATION DES ECHANGES REDOX ENTRE CHLOROPLASTE ET MITOCHONDRIE. EN PLUS DE CET EFFET SUR LES FLUX PHOTOSYNTHETIQUES, L'ABSENCE DU COMPLEXE I A DE FORTES REPERCUSSIONS SUR L'ASSIMILATION DE L'AZOTE. UNE DIMINUTION DANS LA DISPONIBILITE EN 2-OXOGLUTARATE, SQUELETTE CARBONE REQUIS POUR L'ASSIMILATION DE L'AZOTE, EST ASSOCIEE A UNE MODIFICATION DE LA BALANCE C/N, NOTAMMENT UNE ACCUMULATION DES ACIDES AMINES RICHES EN AZOTE. L'ENSEMBLE DES RESULTATS MONTRE QUE L'ETAT REDOX CELLULAIRE EST INFLUENCE PAR LA CHAINE DE TRANSPORT D'ELECTRONS MITOCHONDRIALE, ET SUGGERE QUE L'ETAT REDOX POURRAIT ETRE UN ACTEUR CLE DANS LE CONTROLE DES INTERACTIONS C/N.PLANT DEVELOPMENT AND FUNCTION REQUIRES DIALOGUE BETWEEN DIFFERENT SUBCELLULLAR COMPARTMENTS. INVOLVING EXCHANGE OF METABOLITES, NUCLEOTIDES AND REDUCING POWER. THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN PLAYS AN IMPORTANT PART IN THIS INTERCOMPARTMENTAL CO-ORDINATION. THIS WORK PRESENTS A STUDY OF THE INTEGRATION OF MITOCHONDRIAL PROCESSES IN WHOLE LEAF PHYSIOLOGY AND MET ABOLISM. THE NICOTIANA SYLVESTRIS MUTANT, CMSII (CYTOPLASMIC MALE-STERILE II). IS ONE OF THE FEW AVAILABLE MUTANTS WITH A DYSFUNCTIONAL RESPIRATORY COMPLEX (LOSS OF COMPLEX I ACTIVITY) DUE TO A DELETION IN THE MITOCHONDRIAL GENE, NAD7. THIS MUTANT REPRESENTS, THEREFORE, AN EXCELLENT SYSTEM IN WHICH TO DISSECT THE CONSEQUENCES OF LOSS OF A MAJOR MITOCHONDRIAL DEHYDROGENASE FOR WHOLE LEAF PROCESSES. IT IS SHOWN THAT COMPLEX I DYSFUNCTION CAUSES ADJUSTMENT OF LEAF ANTIOXIDANT SYSTEMS. IMPLICA TING MITOCHONDRIA TO NUCLEUS SIGNALLING IN DETERMINATION OF STRESS RESISTANCE. SECOND, FLUX ANALYSIS OF PHOTOSYNTHESIS DEMONSTRATES THE IMPORTANCE OF COMPLEX I FUNCTION IN OPTIMISING CARBON ASSIMILATION WHEN PHOTORESPIRATION IS ACTIVE AND, PARTICULARLY, DURING THE INDUCTION PERIOD THAT FOLLOWS A DARK/LIGHT TRANSITION. EVIDENCE IS PRESENTED THAT THIS ROLE IS LINKED TO AN OPTIMISATION OF CHLOROPLAST-MITOCHONDRIA REDUCTANT EXCHANGE. IN ADDITION TO THIS EFFECT ON OVERALL PHOTOSYNTHETIC FLUXES, THE ABSENCE OF COMPLEX I HAS DRAMATIC REPERCUSSIONS FOR THE INTEGRATION OF NITROGEN ASSIMILATION WITH CO2 FIXATION. A SUBSTANTIAL MODIFICATION OF LEAF ORGANIC ACID PROFILES INVOLVES DECREASED AVAILABILITY OF 2-OXOGLUTARATE. THE CARBON SKELETON FOR AMMONIA ASSIMILATION. THIS OBSERVATION IS LINKED TO A MARKED CHANGE OF LEAF C/N BALANCE, EVIDENCED BY THE ACCUMULATION OF AMINO ACIDS RICH IN NITROGEN. TAKEN TOGETHER, THE RESULTS INDICATE THAT CELLULAR REDOX STATE IS GREATLY INFLUENCED BY MITOCHONDRIAL ELECTRON TRANSPORT STATUS. AND LEAD TO THE CONCLUSION THAT REDOX STATE MAY BE A KEY PLAYER IN CONTROLLING C/N RELATIONSHIPS.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

l-Galactono-1,4-lactone Dehydrogenase Is Required for the Accumulation of Plant Respiratory Complex I

International audienceMitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation