Le marché mondial des fruits représente des centaines de milliards d’euro par an et l’amélioration de la qualité organoleptique et nutritionnelle des fruits est l’un des principaux objectifs de ces dernières années. Le métabolisme primaire est une cible toute trouvée pour tenter de répondre à ces exigences. En effet c’est lui qui va fournir les briques nécessaires à la croissance et au développement, mais également les composés qui confèrent les valeurs gustatives tels que les sucres et les acides organiques. C’est pourquoi la compréhension de son fonctionnement au cours du développement des fruits est nécessaire. Pour cela le métabolisme primaire a été étudié chez huit espèces de fruits charnus qui diffèrent en termes de durée de développement, de taille de fruit, de famille botanique, de qualité gustative (sucrosité, acidité…), et sujettes ou non à une crise respiratoire au début de la maturation. Des données physiologiques et biochimiques ont été collectées tout au long du développement du fruit, de l’anthèse à la maturité physiologique. La modélisation de la croissance des fruits a permis de standardiser les stades de développement et ainsi d’améliorer la comparaison entre espèces. La composition de la biomasse a ensuite été caractérisée qualitativement et quantitativement par des approches analytiques ciblées et non ciblées mettant en évidence les similitudes et les différences de composition et d’évolution au cours du développement. Des modèles linéaires généralisés combinant la composition et les données de croissance ont été utilisés pour comparer différentes phases de développement du fruit et une analyse discriminante par régression des moindres carrés partiels (PLS-DA) a permis de séparer les fruits climactériques des non climactériques. Dans les deux cas, les composés des parois cellulaires, les protéines et les lipides interviennent dans la différentiation des groupes. Enfin, une étude détaillée du métabolome et de l’activome du fruit a été réalisée chez trois espèces de Solanacées. Elle montre qu’au sein d’une même famille botanique la régulation diffère au cours du développement, notamment au niveau du métabolisme des sucres et de la glycolyse. Ces travaux revisitent le caractère climactérique des fruits, le positionnant bien en amont du déclenchement de la crise respiratoire, et, plus généralement, permettent de mieux comprendre le métabolisme primaire au cours du développement du fruit. / The world fruit market represents hundreds of billions of euros per year and improving the organoleptic and nutritional quality of fruit has been one of the main objectives in recent years. Primary metabolism is a target that can be used to try and meet these requirements. Indeed, it provides the bricks necessary for growth and development but also the compounds that contribute to taste such as sugars and organic acids. Therefore, it is necessary to understand how it operates during fruit development. For this purpose, primary metabolism has been studied in eight species of fleshy fruits that differ in terms of development duration, fruit size, botanical family, taste quality (sweetness, acidity, etc.), and are subject or not to a respiratory crisis at the initiation of ripening. Physiological and biochemical data have been collected throughout the fruit development from anthesis to physiological maturity. Fruit growth modelling allowed standardizing the stages of development and thus improved comparison between species. The composition of biomass was then characterized qualitatively and quantitatively by targeted and non-targeted analytical approaches highlighting similarities and differences in composition and changes during development. Generalized linear models combining composition and growth data were used to compare different phases of fruit development and a discriminant partial least square regression analysis (PLS-DA) was used to separate climacteric and non-climacteric fruits. In both cases, cell wall compounds, proteins and lipids were involved in group differentiation. Finally, a detailed study of the fruit metabolome and activome was performed in three species of Solanaceae. It revealed that within the same botanical family, regulation differs during development, particularly for sugar metabolism and glycolysis. This work revisits the climacteric character of fruits, positioning it long before the onset of the respiratory crisis, and, more generally, provides a better understanding of primary metabolism during fruit development.

Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. Species in this family span the spectrum of host nutrient dependency, allowing comparisons that provide insight into parasite adaptation. A key aspect of this is the relationship between parasite metabolism and the metabolite profile of its host. To what extent does the metabolite profile of the parasite depend on that of the host? Do parasites that differ in host-dependency also differ in their metabolism or do they use common metabolic strategies? These questions were addressed using comparative profiling of primary metabolites to gain insight into carbon and nitrogen assimilation by the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor. First, metabolite profiles of these parasites and their hosts were compared during the key life stages before and after haustorial attachment. Second, the impact of specific variations in host metabolism was analyzed for P. aegyptiaca growing on Arabidopsis thaliana hosts that had mutations in amino acid metabolism but otherwise identical genetic backgrounds. Comparison of P. aegyptiaca and T. versicolor metabolite profiles identified substantial differences in the stages spanning the transition from pre-haustorial development through post-haustorial feeding. Each parasite species is distinct from the other and from their hosts. For parasites growing on host lines that differ in amino acid content, the size of P. aegyptiaca tubercles decreased when grown on the aap6 mutant line, which has decreased levels of asparagine in the phloem sap compared to the wild type. However, altered amino acid levels in other lines did not impact P. aegyptiaca growth, indicating that this parasite has ability to compensate for variation in host metabolic composition. This research highlights the importance of aspartate and asparagine to early post-attachment metabolism in both P. aegyptiaca and T. versicolor and through host deficiencies possibly associated with decreased growth in P. aegyptiaca. Overall, this work provides insights both into the metabolism of parasitic plants and lays the foundation for the development of new metabolism-based control strategies. / Ph. D. / Parasitic weeds of the plant family Orobanchaceae attach to the roots of host plants via haustoria. Parasite haustoria embed into the host plant and are capable of drawing nutrients from host vascular tissue. Species in this family span the spectrum of the extent to which a parasitic plant may depend on its host for nutrients. This allows comparisons that provide insight into the ways in which parasites adapt. A key aspect of this is the relationship between the metabolite profile of the parasite and the metabolite profile of the host. To what extent does the metabolite profile of the parasite depend on that of the host? Do parasites that differ in host-dependency also differ in their metabolism or do they use common metabolic strategies? These questions were addressed using comparative profiling of primary metabolites to gain insight into carbon and nitrogen assimilation by the obligate parasite Phelipanche aegyptiaca (which cannot perform photosynthesis) and the facultative parasite Triphysaria versicolor (which can perform photosynthesis). First, metabolite profiles of these parasites and their hosts were compared during the key life stages before and after haustorial attachment. Second, the impact of specific variations in host metabolism was analyzed for P. aegyptiaca growing on Arabidopsis thaliana hosts. These hosts had mutations in enzymes related to amino acid metabolism but otherwise identical genetic backgrounds. Comparison of P. aegyptiaca and T. versicolor metabolite profiles identified substantial differences in the stages spanning the transition from pre-haustorial development through post-haustorial feeding. Each parasite species is distinct from the other and from their hosts. For parasites growing on host lines that differ in amino acid content, the size of P. aegyptiaca tubercles decreased when grown on the aap6 mutant line, which has decreased levels of asparagine in the phloem sap compared to the wild type. However, altered amino acid levels in other lines did not impact P. aegyptiaca growth, indicating that this parasite has ability to compensate for variation in host metabolic composition. Overall, this work provides insights both into the metabolism of parasitic plants and lays the foundation for the development of new metabolism-based control strategies.

The tricarboxylic acid (TCA) cycle is one of the central pathways in respiration and also plays an important role in a variety of metabolic processes including the synthesis of secondary metabolites and the provision of carbon skeletons for ammonium assimilation and amino acid biosynthesis. Effective regulation of these multiple demands on the TCA cycle is likely to be very important for plant fitness. One way that this regulation could be achieved is through metabolite channelling. This occurs when metabolites are transferred between enzyme active sites without diffusing into the bulk aqueous phase of the cell, and is known to be important in regulating demands in metabolic pathways. Although there is evidence that metabolite channelling exists in animals, there have been no attempts to investigate it in plant. The first aim of this thesis was therefore to investigate whether metabolite channelling exists in the plant TCA cycle. Isotope dilution experiments were developed to investigate metabolite channelling, and were able to show that metabolite channelling was present between certain enzymes of the TCA cycle in both S. tuberosum and A. thaliana mitochondria. The second aim of the thesis was investigate whether metabolite channelling is important in regulating the TCA cycle in plant mitochondria. The pattern of metabolite channelling did not change in mitochondria isolated from the light and the dark, or from mitochondria with increased or decreased TCA cycle rates, but it was not possible to say whether the metabolite channelling altered in a quantitative fashion. Overall the thesis provides the first direct evidence of channelling in the TCA cycle in plants, and further work should help to elucidate what role, if any, it plays.

O uso da metabolômica ambiental tem sido usada para avaliar a interação dos organismos com o ambiente. Apesar do alto impacto que os metais têm no ambiente, essa abordagem analítica ainda está em seu início, em especial para as macroalgas. Como membro do primeiro nível trófico da cadeia alimentar marinha, fornecendo nutrientes e microelementos para os níveis superiores, as macroalgas são um alvo apropriado tanto para o desenvolvimento de ensaios toxicológicos quanto como bioindicador de degradação do ambiente marinho. Também por causa da sua posição na cadeia alimentar, essas macrófitas são consideradas o principal vetor para a magnificação desses elementos tóxicos. Os efeitos tóxicos dos metais sobre o ambiente aquático são documentados e bem conhecidos, mas relacionam principalmente ao desbalanço do potencial redox intracelular e, assim, o estresse oxidativo em organismos vivos. Com o objetivo de compreender a relação das macroalgas com o metal essencial Cu2+ e o metal não essencial Cd2+, a macroalga vermelha Gracilaria domingensis foi selecionada. Após 48h de exposição aos metais em águas do mar sintética e natural, as amostras foram extraídas e analisadas em cromatografia gasosa acoplada à espectrometria de massas. Em seguida, os dados foram pré-processados e pré-tratados para serem utilizados nas análises estatísticas multivariadas (AEM) de PCA e OPLS-DA. A G. domingensis exposta aos metais em água do mar sintética não foram significativamente influenciadas, como indicado pela MVA e pela análise de vias. Apesar disso, alterações significativas foram observadas na exposição aos metais em água do mar natural. Os principais resultados para o Cu2+ foram a interação do metabolismo de glicina, serina e treonina com o metabolismo de glioxilato e dicarboxilato. Foi sugerido que a macroalga poderia estar alterando o modo de adquirir carbono para uma via não fotossintetizante, uma vez que essa via está prejudicada na exposição ao metal. Também, o metabolismo de fenilalanina foi impactado por essa exposição, uma vez que é uma via fundamental para sintetizar compostos fenólicos antioxidantes. Por outro lado, apesar de oito vias terem sido identificadas como significativamente alteradas na exposição ao Cd2+, somente o metabolismo de arginina e prolina parece ter sido significativamente influenciado com o objetivo de produzir prolina, um aminoácido reconhecido por suas propriedades antioxidantes e protetoras em organismos estressados por metais. Em conclusão, os metais essenciais e não essenciais parecem ter mecanismos diferentes na tentativa de promover o combate aos danos gerados pela exposição aos metais. / The environmental metabolomics approach has been used to evaluate the interaction of organisms with their environment. Besides the high impact metals have on the environment, this method of analysis is still in its infancy, in special for macroalgae. As members of the first trophic level in the marine food chain, providing nutrients and microelements to upper levels, macroalgae are appropriate target organisms both for the development of toxicological assays and as a bioindicator of marine degradation. Also, because of their marine food chain position, these macrophytes are considered the main vectors to magnify these toxic elements. The toxic effects of metals on the aquatic environment are documented and well known, but regards mainly to the unbalance of intracellular redox potential and, therefore, oxidative stress in living organisms. In order to understand the relationship of macroalgae with the essential metal Cu2+ and the non-essential metal Cd2+, the red macroalga Gracilaria domingensis was chosen. After 48h of metal exposure in synthetic and natural seawater, the samples were extracted and analysed in gas chromatograph coupled to mass spectrometry. Afterward, the data were preprocessed and pretreated for the multivariative analysis (MVA) with PCA and OPLS-DA statistics. G. domingensis exposed to metals in synthetic seawater were not significantly affected, as indicated by MVA and pathway analysis. Though, significant changes were observed on exposure to metals in natural seawater. The main results for Cu2+ were the interlay of glycine, serine and threonine metabolism with glyoxylate and dicarboxylate metabolism. It was suggested that the macroalga could be shifting the metabolism to acquire carbon from a non-photosynthetic pathway, since it is injured on metal exposure. Also, phenylalanine metabolism was impacted by this metal exposure, since it is a pivotal source of phenolic antioxidant compounds. On the other hand, besides eigth pathways were identified as significantly changed on Cd2+ exposure, only arginine and proline metabolism seemed to be significantly affected, in order to produce proline, known for its antioxidative and protective properties in metal stressed organims. In conclusion, essential and non essential metals seem to have distinct mechanism to mitigate the damaged caused by metal exposure.

O uso da metabolômica ambiental tem sido usada para avaliar a interação dos organismos com o ambiente. Apesar do alto impacto que os metais têm no ambiente, essa abordagem analítica ainda está em seu início, em especial para as macroalgas. Como membro do primeiro nível trófico da cadeia alimentar marinha, fornecendo nutrientes e microelementos para os níveis superiores, as macroalgas são um alvo apropriado tanto para o desenvolvimento de ensaios toxicológicos quanto como bioindicador de degradação do ambiente marinho. Também por causa da sua posição na cadeia alimentar, essas macrófitas são consideradas o principal vetor para a magnificação desses elementos tóxicos. Os efeitos tóxicos dos metais sobre o ambiente aquático são documentados e bem conhecidos, mas relacionam principalmente ao desbalanço do potencial redox intracelular e, assim, o estresse oxidativo em organismos vivos. Com o objetivo de compreender a relação das macroalgas com o metal essencial Cu2+ e o metal não essencial Cd2+, a macroalga vermelha Gracilaria domingensis foi selecionada. Após 48h de exposição aos metais em águas do mar sintética e natural, as amostras foram extraídas e analisadas em cromatografia gasosa acoplada à espectrometria de massas. Em seguida, os dados foram pré-processados e pré-tratados para serem utilizados nas análises estatísticas multivariadas (AEM) de PCA e OPLS-DA. A G. domingensis exposta aos metais em água do mar sintética não foram significativamente influenciadas, como indicado pela MVA e pela análise de vias. Apesar disso, alterações significativas foram observadas na exposição aos metais em água do mar natural. Os principais resultados para o Cu2+ foram a interação do metabolismo de glicina, serina e treonina com o metabolismo de glioxilato e dicarboxilato. Foi sugerido que a macroalga poderia estar alterando o modo de adquirir carbono para uma via não fotossintetizante, uma vez que essa via está prejudicada na exposição ao metal. Também, o metabolismo de fenilalanina foi impactado por essa exposição, uma vez que é uma via fundamental para sintetizar compostos fenólicos antioxidantes. Por outro lado, apesar de oito vias terem sido identificadas como significativamente alteradas na exposição ao Cd2+, somente o metabolismo de arginina e prolina parece ter sido significativamente influenciado com o objetivo de produzir prolina, um aminoácido reconhecido por suas propriedades antioxidantes e protetoras em organismos estressados por metais. Em conclusão, os metais essenciais e não essenciais parecem ter mecanismos diferentes na tentativa de promover o combate aos danos gerados pela exposição aos metais. / The environmental metabolomics approach has been used to evaluate the interaction of organisms with their environment. Besides the high impact metals have on the environment, this method of analysis is still in its infancy, in special for macroalgae. As members of the first trophic level in the marine food chain, providing nutrients and microelements to upper levels, macroalgae are appropriate target organisms both for the development of toxicological assays and as a bioindicator of marine degradation. Also, because of their marine food chain position, these macrophytes are considered the main vectors to magnify these toxic elements. The toxic effects of metals on the aquatic environment are documented and well known, but regards mainly to the unbalance of intracellular redox potential and, therefore, oxidative stress in living organisms. In order to understand the relationship of macroalgae with the essential metal Cu2+ and the non-essential metal Cd2+, the red macroalga Gracilaria domingensis was chosen. After 48h of metal exposure in synthetic and natural seawater, the samples were extracted and analysed in gas chromatograph coupled to mass spectrometry. Afterward, the data were preprocessed and pretreated for the multivariative analysis (MVA) with PCA and OPLS-DA statistics. G. domingensis exposed to metals in synthetic seawater were not significantly affected, as indicated by MVA and pathway analysis. Though, significant changes were observed on exposure to metals in natural seawater. The main results for Cu2+ were the interlay of glycine, serine and threonine metabolism with glyoxylate and dicarboxylate metabolism. It was suggested that the macroalga could be shifting the metabolism to acquire carbon from a non-photosynthetic pathway, since it is injured on metal exposure. Also, phenylalanine metabolism was impacted by this metal exposure, since it is a pivotal source of phenolic antioxidant compounds. On the other hand, besides eigth pathways were identified as significantly changed on Cd2+ exposure, only arginine and proline metabolism seemed to be significantly affected, in order to produce proline, known for its antioxidative and protective properties in metal stressed organims. In conclusion, essential and non essential metals seem to have distinct mechanism to mitigate the damaged caused by metal exposure.

Orientador: Carmen Silvia Fernandes Boaro / Resumo: Espécies vegetais são capazes de produzir diversidade de substâncias, que desempenham funções importantes para sua sobrevivência e adaptação ao ecossistema. O metabolismo primário, é essencial para o crescimento, desenvolvimento, maturação e reprodução de qualquer espécie. O metabolismo especializado, dependente do primário, é responsável por originar o óleo essencial, que são misturas de metabólitos especializados voláteis, representados principalmente por monoterpenos e sesquiterpenos. Cada espécie vegetal produz um óleo essencial de composição característica específica, podendo ser influenciado por fatores bióticos e abióticos. A fenologia pode influenciar processos bioquímicos e rotas metabólicas capazes de modificar a formação de substâncias biologicamente ativas, alterando diretamente o conteúdo e a qualidade dos óleos essenciais. Sendo assim, o objetivo deste trabalho foi avaliar se as fases fenológicas, vegetativa e reprodutiva modificam o desempenho fotossintético e o perfil do óleo essencial de Xylopia aromatica (Lam.) Mart., influenciando sua atividade biológica na defesa antioxidante e ação antifúngica. As variáveis, fluorescência da clorofila a, trocas gasosas, carboidratos, atividade enzimática e peroxidação lipídica, potencial água, conteúdo relativo de água das folhas, extração, rendimento, caracterização química e atividade antifúngica do óleo essencial de Xylopia aromatica foram avaliadas em 24 plantas, 12 no estádio vegetativo e 12 no reprodutivo, coletadas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Research aimed at the knowledge of plant species allows the elaboration of projects that aim at the understanding of development, conservation of biodiversity and sustainable exploitation of natural resources. The primary metabolism, represented by photosynthesis and the specialized one, that synthesizes the essential oil, can be influenced by the environmental and phenological conditions, which can influence the chemical profile of the essential oil and the biological activity in the vegetal defense, including against fungi, bacteria and virus. Compounds from the specialized metabolism present biological activity and potential for the production of bactericides and fungicides. Therefore, it is necessary to know the stage of development of plant species in which the substances of interest, with economic potential, are more concentrated, thus orienting, if appropriate, the collection period, aiming at the conservation and sustainable use. There are scientific studies that reveal biological activity of essential oils, as observed for the genus Xylopia, but none of them relates the primary and specialized metabolism to the stage of development in which the species is found. In this way, the objective of this research was to evaluate if the phenological, vegetative and reproductive phases of Xylopia aromatica (Lam.) Mart. modify the photosynthetic performance and the profile of the essential oil, which may influence its biological activity in the antioxidant defense and antifunga... (Complete abstract click electronic access below) / Mestre

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Les plantes sont des organismes immobiles qui doivent répondre et s’adapter à des contraintes abiotiques et biotiques. Parmi les stress biotiques, les maladies virales, établies ou émergentes, peuvent être responsables de pertes de rendement majeures aux conséquences économiques importantes. Face aux phytovirus la lutte génétique constitue le moyen de lutte le plus efficace, le plus respectueux de l’environnement et du consommateur. Comprendre l’interaction entre les plantes et les virus reste indispensable pour rechercher de nouvelles sources de résistances. Ce travail de thèse s’intéresse à l’étude du pathosystème naturel Arabidopis thaliana/Turnip mosaic virus (TuMV). Les essais ont été menés majoritairement en conditions extérieures permettant une analyse de l’interaction dans un environnement multistress. La réponse d’A. thaliana a été explorée par l’étude de traits liés à la maladie et par la variation en métabolites primaires et secondaires. Ce travail a permis i) de caractériser de façon fine la réponse d’A. thaliana au TuMV en conditionsmultistress en exploitant la diversité naturelle d’une population mondiale et française ii) de déterminer l’architecture génétique de cette interaction par des approches de génétique d’association et de QTL mapping. Plusieurs nouveaux loci potentiellement impliqués dans la réponse ont été identifiés iii) de montrer l’intérêt du phénotypage métabolique pour discriminer les accessions en fonction de leur sensibilité au TuMV. La multidisciplinarité des approches constitue la richesse de ce travail de thèse qui contribue à une meilleure caractérisation et compréhension de la réponse des plantes lors d’une infection virale. / Plants are immobile organisms which have to adapt to abiotic and biotic constraints. Among bioticstress, established or emerging viral diseases, may be responsible for major yield losses withsignificant consequences. Genetic control is the most effective, environmentally and consumerfriendlyway to control viral infections. Understanding plant/virus interactions remains essential tosearch for new sources of resistance. This work, focuses on the study of the natural pathosystemArabidopsis thaliana/Turnip mosaic virus (TuMV). Most of the trials were conducted in commongarden conditions allowing the analysis of the interaction in a multistress environment. A. thaliana’sresponse was explored through the study of disease-related traits and the variations in primary andsecondary metabolites. This work allows i) the fine characterization of A. thaliana’s response toTuMV in multistress conditions through the exploration of the natural diversity of a world and Frenchpopulation ii) to determine the genetic architecture of this interaction by genome wide associationsand QTL mapping. Several new loci potentially involved in the response have been identified iii) tohighlight the interest of metabolic phenotyping to discriminate accessions according to theirsusceptibility to TuMV. The multidisciplinary approaches contribute to a better characterization andunderstanding of plant-virus interaction.

Les insectes endophytophages, tels que les insectes foreurs de tiges, les galligènes et les mineurs de feuilles, vivent et se nourrissent à l’intérieur des végétaux. L'hypothèse de l'alimentation sélective stipule que ces organismes endophytes possèdent un avantage adaptatif par rapport aux ectophages en accédant aux tissus les plus nutritifs tout en évitant les principaux composés défensifs de la plante. Ce comportement d’alimentation sélective peut être également renforcé par une manipulation de la physiologie de la plante comme cela a été démontré chez les insectes galligènes mais également suggéré chez certains insectes mineurs. Ces derniers sont en effet capables d’induire un phénotype « îles vertes » qui se manifestent par la persistance de la photosynthèse au niveau de la zone minée à l'automne alors que le reste de la feuille entre en sénescence et jaunit. L’objectif de notre étude a été d’étudier, en conditions de terrain, les capacités de manipulation du végétal dans le système Malus domestica / Phyllonorycter blancardella. Cet insecte hautement spécialisé complète l’ensemble de son développement dans une zone restreinte d’une seule feuille. / Endophytophagous insects, such as stem-boring, gall-forming and leaf-mining insects, live within plant tissues and feed internally. The selective feeding hypothesis states that this life-style presumably provides adaptive advantages for the insect over other external-feeding modes by allowing access to most nutritional tissues while avoiding main plant defensive compounds. This selective feeding behavior can be reinforced by manipulating the plant physiology which has been clearly demonstrated in gallers but also suggested in leaf-miner insects due to the autumnal formation of “green islands” around mining caterpillars in yellow leaves. This study aimed to investigate, under field conditions, the ability of insects to manipulate their host-plant in the Malus domestica / Phyllonorycter blancardella biological system. This insect is highly specialized and entirely develops within a restricted area of a single leaf. We first characterized the plant-insect interface by describing larval mouthparts and leaf anatomy alterations resulting from the insect feeding activity.