<p>C₄-dicarboxylic acids appear to be metabolized via the TCA cycle in N₂-fixing bacteria (bacteroids) within legume nodules. In <em>Sinorhizobium meliloti</em> bacteroids from alfalfa, NAD⁺-malic enzyme (DME) is required for symbiotic N₂-fixation and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont <em>Rhizobium leguminosarum</em> pyruvate synthesis can occur via either the DME pathway or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK), pyruvate kinase (PYK), and pyruvate dehydrogenase. Here we report that <em>dme</em> mutants of <em>Sin</em>or<em>hizobium sp</em>. NGR234 formed root nodules on a broad range of plants and that the level of N₂-fixation varied from 90% to 20% of wild type depending on the host plants inoculated. NGR234 bacteroids had significant PCK activity and while single <em>pckA</em> and single <em>dme</em> mutants fixed N₂ on <em>Macroptilium atropurpureum</em> and <em>Leucaena leucocephala</em> (albeit at a reduced rate), a <em>pckA</em> <em>dme</em> double mutant had no N₂-fixing activity (Fix^-). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix^- phenotype of <em>S. meliloti dme </em>mutants may be specific to the alfalfa-<em>S. meliloti </em>symbiosis. We therefore examined the ME-like genes <em>azc3656 </em>and <em>azc0119 </em>from <em>Azorhizobium caulinodans</em>, as <em>azc3656 </em>mutants were previously shown to form Fix^- nodules on the tropical legume <em>Sesbania rostrata</em>. We found that purified AZC3656 protein is an NAD (P)⁺-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N₂ fixation in <em>A. caulinodans </em>and <em>S. meliloti</em>, in other rhizobia this activity can be bypassed via another pathway(s).</p> <p>In <em>S. meliloti</em> both malic enzymes DME and TME share similar apparent <em>K_m</em>s for substrate and cofactors, but differ in their responses to TCA cycle intermediates, with DME activity inhibited by acetyl-CoA and induced by succinate and fumarate. Previous results in our laboratory indicated that DME is essential for symbiotic N₂ fixation, while TME fails to functionally replace DME. One possible reason for it is that a high ratio of NADPH/NADP⁺in<em> S. meliloti </em>bacteroids prevents TME from functioning in nodules. We sought to lower the<em> </em>NADPH/NADP⁺ratio by overexpressing a soluble pyridine nucleotide transhydrogenase (STH). However, metabolite measurements indicated that overproducing STH failed to lower the ratio of NADPH/NADP⁺ in<em> S. meliloti</em>.</p> <p>Previous studies assumed that DME and TME might play different roles in central carbon metabolism. To gain insight of their physiological functions, genome-wide microarray analysis was conducted in <em>S. meliloti</em> single<em> dme and</em> <em>tme</em> mutants grown on glucose or succinate. The most striking changes of gene expression were observed in <em>S. meliloti</em> <em>dme</em> mutants grown on succinate. The functions of upregulated genes suggested that DME might play an important role in regulating TCA cycle intermediates, important for the maintenance of metabolic flux through TCA cycle during C₄-dicarboxylate oxidation. However, changes of gene expression found in <em>tme </em>mutants were not significant enough to predict the physiological functions of TME protein in central carbon metabolism.</p> / Doctor of Philosophy (PhD)

Immune systems are fundamentally based on the differentiation of self and non-self. Unlike mammals, plants have an innate immune system responding to the pathogen only at the site of attack. One of these pathogens is Blumeria graminis f. sp. hordei which is an obligate biotrophic pathogen causing powdery mildew disease and resulting in up to 30% yield loss for both cultivated and wild barley. In this study, Pallas-01 (P-01) and Pallas-03 (P-03) barley lines were inoculated with powdery mildew race Bgh103 (64/01) resulting incompatible and compatible interactions, respectively. 6, 12, 24, 48 and 72 hour-post-inoculation (hpi) samples were used in order to define the differential gene expression of NAD malic enzyme, chloroplast lipocalin, phosphoglyceromutase (PGM), Mg chelatase and 26S protease regulatory subunit 6B homolog. In the proteomics study previously conducted in the laboratory, except for the NAD-dependent malic enzyme, the other four proteins were shown to be involved in the incompatible interaction of P-01 and Bgh103 at protein level, whereas NAD-dependent malic enzyme was changing in the compatible interaction. The expression level for both proteomics and transcriptomics were assumed to be similar. However, the level of transcript would not always reflect its protein level or correlate with the level of proteins, due to complex cellular regulation processes. Post-transcriptional modifications such as synthesis, processing, degradation and post-translational modifications are changing the level of proteins expressed, thus a parallel correlation between the protein and mRNA levels can be lost. Other possible reasons for this variation can be changes in mRNA and protein stability, efficiency of translation and protein&rsquo / s turnover rate. The transcription level changes of the genes investigated in this study are found to be differentially expressed, supporting the proteomics data indicating that these genes are possibly involved in resistance. For further investigations, genetic tools such as gene silencing with RNAi and knockout experiments are required in order to elucidate the mechanism of these candidate genes in the plant-pathogen interaction.

Stomata are small pores that are located on the surface of epidermal leaves, and they can regulate the uptake of CO2 and prevent water lose by opening and closing the pores. Stomata of plants can be regulated by external condition such as CO2, biotic and abiotic stresses and internal factors. CAM (crassulacean acid metabolism) plants adapt to hot and dry environments by closing stomata during the day and opening stomata during the cool night. However, it is still unclear how CAM plants open their stomata during the night and close them during the day. In this study, a number of factors were evaluated for their potential roles in promoting stomatal opening in the model CAM plant Kalanchoe laxiflora. Citrate is an important organic acid and it accumulates during the night in CAM plants. It is shown in this study that citrate promoted stomatal opening in detached leaf epidermis of Kalanchoe laxiflora. Further, the cytokinin zeatin is also shown to stimulate stomatal opening in detached leave of Kalanchoe laxiflora. Melatonin is an important regulator of circadian rhythms in mammals and has been implicated in regulation of plant abiotic stress responses. Melatonin was detected in the leaves of Kalanchoe laxiflora. It promoted stomatal opening in detached epidermis of Kalanchoe laxiflora. Together, these results suggest that stomata of Kalanchoe laxiflora respond to citrate and malate which are the main organic acids accumulate during nighttime and also to some signaling molecules (zeatin, melatonin, and serotonin) by opening stomata during dark period.

Le stress oxydant est un acteur clé dans les réponses des plantes à des conditions contraignantes. En raison de la complexité de la régulation de l’état redox cellulaire, il reste beaucoup à élucider concernant les interactions entre différentes composantes dans ces conditions. Grâce à une approche de génétique inverse basée sur un mutant d’Arabidopsis déficient en catalase (cat2) qui présente des modifications d’état redox prévisibles et bien définies, cette étude a exploré les interactions entre le stress oxydant et (1) un gène spécifique impliqué dans la déphosphorylation des protéines, (2) des enzymes spécifiques impliquées dans les systèmes antioxydants réducteurs. Les résultats obtenus révèlent que la sous-unité B'γ de la protéine phosphatase de type 2A (PP2A-B'γ) est importante dans la détermination des phénotypes et des réponses de défense photopériode-dépendantes chez cat2. En conditions de jours courts (SD), un double cat2 pp2a-b'γ mutant montrait une gamme de réponses qui n’étaient pas observées chez cat2. Ces effets comprenaient l’apparition de lésions ainsi que l’accumulation de l’acide salicylique et d’autres composés de défense. Des analyses métabolomiques et protéomiques ont permis de démontrer que ces effets étaient accompagnés de modifications de l’abondance de métabolites et protéines spécifiques, ainsi que des changements dans le statut de phosphorylation de certains polypeptides. Dans un deuxième volet du travail, l’importance d’une enzyme productrice du NADPH a été évaluée en produisant des doubles cat2 nadp-me2 mutants chez lesquels l’isoforme majeure de l’enzyme malique cytosolique n’est plus exprimée. Malgré une induction de cette enzyme par le stress oxydant aux niveaux de transcrits et d’activité, et une diminution importante de l’activité foliaire associée aux mutations nadp-me2, peu de différence a été observée entre les lignées cat2 et cat2 nadp-me2. De même, la mutation nadp-me2 n’a pas affecté la réponse phénotypique de plantes exposées à l’ozone. Dans la troisième partie du travail, le couplage entre les pools ascorbate et glutathion lors du stress oxydant a été exploré par l’introduction de mutations pour la déshydroascorbate réductase (DHAR) dans le fond génétique cat2. L’activité extractible de cette enzyme a été diminuée à des niveaux très faibles chez des lignées portant à la fois les mutations dhar1 et dhar3. Cependant, peu de différence a été observée dans les phénotypes et les statuts d’ascorbate et de glutathion chez un triple mutant cat2 dhar1 dhar3 par rapport à cat2. Des analyses préliminaires d’un quadruple cat2 dhar1 dhar2 dhar3 mutant semblent pourtant indiquer que les trois DHARs jouent des rôles fonctionnellement redondants dans le stress oxydant. Dans son ensemble, ces travaux apportent des données nouvelles sur les enzymes qui régulent les réponses aux stress oxydants et ont généré des outils intéressants pour des études ultérieures. / Oxidative stress is a key player in plant responses to challenging environmental conditions. The intricate nature of the regulation of cellular redox state means that much remains to be elucidated on interactions between different components in these conditions. By using a genetic approach based on a catalase-deficient Arabidopsis mutant (cat2) that presents well-defined, predictable changes in redox state, this study explored interactions between oxidative stress and (1) a specific gene involved in protein dephosphorylation, and (2) specific enzymes involved in the antioxidative/reducing system. The results showed that protein phosphatase 2 subunit B'γ (PP2A-B'γ) is involved in determining day length-dependent phenotypes and related defense responses in cat2. A cat2 pp2A-B'γ double mutant showed a range of responses that were not observed in cat2 grown in short days, including lesion formation and accumulation of salicylic acid (SA) and related metabolites. Metabolomics and proteomics analyses showed that these effects were associated with altered abundance of specific metabolites and proteins, as well as changes in protein phosphorylation status. A second part of the study investigated the importance of NADP-generating enzymes in oxidative stress by production of cat2 nadp-me2 double mutants, in which the cytosolic isoform of NADP-malic enzyme is knocked out. Although NADP-ME2 was shown to be induced by oxidative stress, and mutants for this gene had much decreased leaf NADP-malic enzyme activity, no effects on cat2 phenotypes or redox profiles were apparent. Similarly, phenotypic responses to ozone were not affected in an nadp-me2 single mutant. In the third part, coupling between ascorbate and glutathione pools during oxidative stress was investigated by introduction of loss of function mutations for dehydroascorbate reductase (DHAR) into the cat2 background. In lines carrying a combination of dhar1 and dhar3 mutations, extractable leaf activity was decreased to very low levels. Despite this, cat2 dhar1 dhar3 and cat2 phenotypes and ascorbate and glutathione pools were similar. However, preliminary functional analysis of a cat2 dhar1 dhar2 dhar3 quadruple mutant suggested that the three DHARs play functionally redundant roles in oxidative stress. Overall, the work provides new data on enzymes that regulate responses to oxidative stress and has produced interesting genetic tools for further study.

Biotic stress in the form of viral infection, as well as abiotic salt stress, cause leaves injuries, stomata closure and decreased rate of photosynthesis. These factors lead to the limitation of plant growth and to reduced amount of coenzyme NADPH. However NADPH is an important coenzyme for many metabolic pathways such as synthesis of fatty acids, amino acids and secondary metabolites involved in stress responses. NADPH is also a coenzyme for key enzymes of antioxidant system and for many regulatory enzymes. NADP-dependent enzymes are alternative source of NADPH in plants under stress conditions. In this work, activities of four NADP-dependent enzymes: Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), NADP-malic enzyme (decarboxylating) (NADP-ME, EC 1.1.1.40) and Shikimate dehydrogenase (SDH, EC 1.1.1.25) were studied. Activities of all these enzymes but SDH increased in leaves of tobacco plants (Nicotiana tabacum L.) infected by PVYNTN , The most sensitive enzymes to viral infection were NADP-ICDH and NADP-ME, whose activity was increased in comparison with control plants 3-fold and 2,4-fold, respectively. Changes in activity of studied enzymes were also determined in plants exposed to viral infection in combination with heat-shock...