Chlorophyll fluorescence imaging is noninvasive technique often used in plant physiology, molecular biology and precision farming. Captured sequences of images record the dynamic of chlorophyll fluorescence emission which contain the information about spatial and time changes of photosynthetic activity of plant. The goal of this Ph.D. thesis is to contribute to the development of chlorophyll fluorescence imaging by application of advanced statistical techniques. Methods of statistical pattern recognition allow to identify images in the captured sequence that are reach for information about observed biotic stress and to find small subsets of fluorescence images suitable for following analysis. I utilized only methods for identification of small sets of images providing high performance with realistic time consumptions.

La compréhension des mécanismes de défense mis en place lors de la résistance des plantes vis-à-vis d'agents pathogènes a pour objectif de proposer des alternatives à l'utilisation de produits phytosanitaires utilisés en agriculture. Dans une première partie, nous avons étudié les mécanismes moléculaires impliqués dans la résistance induite aux pathogènes par l'acide β-aminobutyrique (BABA) chez la vigne. En effet, cet acide aminé non protéique favorise un état physiologique particulier, appelé potentialisation, dans lequel la plante est capable de mobiliser plus rapidement et/ou plus intensément ses réactions de défense en réponse à un stress. Contrairement aux éliciteurs comme les oligogalacturonates (OG), nous avons montré que le BABA seul n’induisait pas les événements précoces de signalisation sur suspensions cellulaires de vigne, tels que les variations de la concentration en calcium cytosolique libre ([Ca2+]cyt), la production de monoxyde d’azote (NO), la production d’H2O2, la phosphorylation de MAPkinases, ni l’expression de gènes de défense. Seules la production d’H2O2 et l’expression plus intense du gène RbohD codant une NADPH oxydase sont potentialisées par le BABA dans les suspensions cellulaires élicitées par les OG. In planta, le BABA potentialise également une production d’H2O2 en réponse à l’infection par l’oomycète Plasmopara viticola. L’utilisation d’un inhibiteur de NADPH oxydase abolit complètement cette production d’H2O2 et bloque partiellement la résistance induite par le BABA. Nous montrons donc que la potentialisation de la production d’H2O2 dépendante d’une NADPH oxydase contribue à l’établissement de la résistance induite par le BABA chez la vigne. Une deuxième partie a permis d’appréhender les événements cellulaires impliqués dans la résistance des plantes en se focalisant sur le mutant pad2 (phytoalexin deficient) d’Arabidopsis thaliana. Ce mutant présente une sensibilité accrue à différents pathogènes et contient un taux de glutathion de l’ordre de 20 % par rapport à l’écotype sauvage. Nous avons tout d’abord montré que le faible taux de glutathion dépendait d’une quantité réduite de la première enzyme de sa biosynthèse, la glutamate-cystéine ligase. Le glutathion étant impliqué dans la mise en place des réactions de défense, nous avons tenté de définir le lien entre la déficience en glutathion et la sensibilité de pad2 aux pathogènes. Nous avons tout d’abord montré que pad2 possédait un état redox du glutathion plus oxydé que le sauvage. Une analyse transcriptomique à l’état basal a révélé que la plupart des gènes différentiellement exprimés étaient réprimés chez pad2. Parmi ces gènes, certains codent des protéines impliquées dans les flux d’ions qui pourraient déréguler la dépolarisation membranaire. Nous avons ainsi confirmé que la dépolarisation de la membrane plasmique est amoindrie chez pad2 en réponse aux OG. De plus, des événements en aval tels que la production d’H2O2 et la production de NO sont également plus faibles chez le mutant par rapport au sauvage. Cette absence de la production d’H2O2 a également été spécifiquement observée sur plantes pad2 infectées par l’oomycète Phytophthora brassicae. Il en résulte un développement accru du pathogène corrélé à une absence de réponse hypersensible, une mort cellulaire localisée normalement observée dans le cas du sauvage résistant. En réponse aux OG ou à l’infection par P. brassicae, les analyses transcriptomiques font ressortir un fort enrichissement de gènes relatifs à la dégradation des protéines chez pad2. De manière globale, nos résultats suggèrent que la déficience en glutathion chez pad2 pourrait profondément modifier le turn-over des protéines, perturbant ainsi la signalisation cellulaire et les réponses biologiques associées. / Alternative strategies are required to reduce pesticide input into the environment for effective and sustainable plant protection. One solution is the activation of plant basal resistance that relies on the application of resistance inducer molecules. In the first part of this study, we analyzed the mode of action of β-aminobutyric acid (BABA), a non-protein amino acid, in the grapevine induced resistance. BABA confers a physiological state, called priming, during which plants are able to mobilize better and/or more rapidly defense responses to biotic or abiotic stress. Unlike oligogalacturonides (OG), we showed that BABA did not induce early signaling events in grapevine cells such as variations of cytosolic free calcium concentration, H2O2 and nitric oxide production, MAPkinase phosphorylation, nor the expression of defense-related genes. Among them, only H2O2 production and the expression of RbohD gene, which encodes a NADPH oxidase, are primed by BABA in OG-treated cells. Moreover, BABA-treated plants display a stronger accumulation of H2O2 in response to the oomycete Plasmopara viticola. Application of an NADPH oxidase inhibitor completely abolishes this H2O2 production and leads to a reduction of BABA-induced resistance against P. viticola. These data suggest that the priming of an NADPH oxidase-dependent H2O2 production contributes to BABA-induced resistance in grapevine. The second part consisted to analyze molecular events involved in plant resistance by using the pad2 (phytoalexin deficient) mutant of Arabidopsis thaliana which is susceptible to a broad range of pathogens. We showed that the glutathione depletion depends on the low amount of glutamate-cysteine ligase protein, the first enzyme involved in its biosynthesis. We studied molecular events, which are involved in defense mechanisms, to understand the impact of the glutathione content on pad2 susceptibility. Our results show that the redox state of glutathione is more oxidized in pad2 than in wild type Col-0. Since cellular redox state change is known to regulate gene expression, a basal transcriptome analysis has been performed in pad2 and wild type plants. Interestingly, most of the identified genes in pad2 are down-regulated, some of them encoding proteins involved in ion fluxes. As expected, the plasma membrane depolarization and events downstream like H2O2 and NO production are impaired in pad2 in response to OG. During infection with Phytophthora brassicae, the lack of H2O2 production is concomitant with an absence of the hypersensitive response, a localize cell death observed in the resistant wild type. After OG treatment or P. brassicae infection, microarray analysis brings out genes related to protein machinery including degradation in pad2. Taken together, these data suggest that the depletion of glutathione has an impact on protein turn-over which disturbs cell signaling events and related biological responses.

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...

Auto-fluorescence emission of plant tissues can be a powerful reporter on plant biochemistry and physiology since it originates in substances inherent to primary or secondary metabolism. Plant bodies contain a plethora of intrinsic fluorescent compounds emitting practically all wavelengths of visible light. Moreover, the spectrum of fluorescent reporter signals was recently extended by a variety of fluorescent proteins that provide a new tool to mark whole cells or sub-cellular structures, study protein localization and monitor gene expression and molecule interactions. The imaging of such fluorescence signals reveals a possibility to acquire the information from as many as millions of points simultaneously, in vivo and in a non-invasive way thereby preserving integrity of cells and whole organisms. Imaging is particularly suited to visualize heterogeneity such as a localized immune response to invading pathogens. It can be applied both at macro- as well as micro-scales in two and three dimensions. The recent advancement in microscopy, the multi-photon microscopy, has made possible to monitor fluorescence signals, such as NAD(P)H fluorescence from intact leaf interior, that have been hidden to single-photon techniques.