La formation des fleurs comprend trois étapes successives. Tout d'abord, un méristème, contenant les cellules souches, se forme sur les flancs du méristème d'inflorescence. Puis, le méristème adopte une identité florale. Enfin, la morphogenèse florale permet le développement des différents organes floraux répartis en quatre verticilles. Ces étapes font intervenir des réseaux génétiques distincts. Le facteur de transcription LEAFY (LFY) est un régulateur majeur du développement floral chez les plantes à fleurs. Le but de ma thèse était de comprendre les fonctions précises de LFY au cours du développement floral, en particulier dans les étapes précoces du développement. Les études moléculaires de LFY chez la plante modèle A. thaliana ont permis de montrer que cette protéine a la capacité de se multimériser lors de sa liaison à l'ADN. En étudiant l'importance fonctionnelle de la dimérisation de LFY, j'ai pu mettre en évidence l'importante de cette propriété pour la régulation de ses gènes cibles, responsables de l'identité florale. De plus, en couplant des études génétiques, les études transcriptomiques et les données de liaisons à l'ADN à l'échelle génomique, j'ai mis en évidence un nouveau réseau de gènes régulé par LFY et impliqué dans le développement du méristème, avant sa détermination en fleur. Ces données ouvrent la perspective que cette nouvelle fonction de LFY est une fonction indépendante de sa fonction florale et déjà présente chez la plupart des plantes terrestres.LFY est hautement conservé chez toutes les plantes terrestres, mais ne fait pas partie d'une famille multigénique contrairement à la plupart des facteurs de transcription qui ont formé des familles multigéniques par duplication au cours de l'évolution. J'ai étudié l'évolution des propriétés de LFY, notamment sa capacité de se dimériser. Pour cela, nous nous sommes intéressés aux homologues de LFY et nous avons découvert que LFY était déjà présent chez les algues vertes multicellulaires. En étudiant l'interface de dimérisation chez les différents homologues de LFY, nous avons mis en évidence que l'acquisition de cette propriété a joué un rôle crucial dans l'évolution de la protéine.Enfin, je me suis intéressé au contrôle post-traductionnel de l'activité de la protéine LFY. Les résultats préliminaires sont présentés et permettent de penser que ce mode de régulation est important pour les fonctions de ce facteur de transcription unique. / Flower formation comprises three successive steps. First, a new meristem, containing stem cells, is formed on the flanc of the inflorescence meristem. Then, this meristem adopts a floral identity. Finally, floral morphogenesis occurs that allow the development of floral organs arranged into four distinct whorls. The LEAFY (LFY) transcription factor is a major regulator of floral development in flowering plants. The aim of my thesis was to precisely understand the roles of LFY during floral development, especially during early stages. Previous studies in the model plant A. thaliana demonstrate that LFY can multimerize upon binding to DNA. By studying the functional importance of the dimerization property of LFY, we were able to show that this property is important for the regulation of its target genes, including those responsible for floral identity. In addition, by combining genetic studies, transcriptomic datas as well as whole-genome LFY binding sites, we have shown that LFY controls a new network of genes which are directly involved in meristem formation, before its determination into flower. These data raise the prospect that this new function of LFY is in fact a non-floral function already present in most land plants.LFY is highly conserved in all land plants, but is not part of a multigene family in contrast to most transcription factors. I studied the evolution of LFY properties, including its ability to dimerize on specific DNA sequences. For this purpose, we looked for the ancestor form of LFY and found out that LFY was already present in multicellular green algae. By studying the dimerization interface in different counterparts of LFY, we demonstrate that the acquisition of this dimerization property has played a crucial role during the evolution of the protein.Finally, I studied the post-translational control of LFY activity which remains largely unknown. Preliminary results are presented and suggest that this mode of regulation is important for many functions of this orphan transcription factor.

L'auteur n'a pas fourni de résumé en français / L'auteur n'a pas fourni de résumé en anglais

L’ARN hélicase UPF1 est un facteur clé du Nonsense-Mediated Decay (NMD), un mécanisme impliqué dans le contrôle de la qualité des ARNm et la régulation de l’expression des gènes. Malgré d’importantes fonctions chez les plantes, le NMD y est peu décrit. Cette thèse présente l’identification et l’étude des protéines interagissant avec UPF1 chez Arabidopsis. Nous avons identifié un nouveau réseau d’interaction protéine-protéine entre UPF1 et des répresseurs de traduction dans les P-bodies. Nous proposons un modèle dans lequel la répression traductionnelle exerce une action protectrice sur les cibles du NMD. Notre approche a également identifié de nouveaux composants des P-bodies, comme l’endonucléase UCN. Son étude détaillée a révélé un lien direct avec la machinerie de decapping ainsi que de possibles rôles dans la signalisation hormonale ou les mécanismes de défense, suggérant que la modulation de l’expression d’UCN pourrait influencer d’importantes caractéristiques agronomiques. Ce travail décrit des facteurs associés à UPF1 jusqu’alors inconnus, leur étude permettra de découvrir de nouveaux mécanismes impliqués dans l’équilibre entre la traduction, le stockage et la dégradation des ARNm chez les plantes. / The RNA helicase UPF1 is a key factor of Nonsense-Mediated Decay (NMD), a paneukaryotic mechanism involved in mRNA quality control and fine-tuning of gene expression. Despite important biological functions in plants, NMD is poorly described compared to other eukaryotes. This thesis presents the identification and study of UPF1 interacting proteins in Arabidopsis. Using approaches based on immunoaffinity and mass spectrometry, we identified a novel protein-protein interaction network between UPF1 and translation repressors in P-bodies. We propose a model in which translation repression exerts a protective action on NMD targets in plants. Our approach also identified novel P-body components, including the UCN endonuclease. A detailed study revealed its direct link with the decapping machinery and possible roles in hormone signaling and defense mechanisms, suggesting that the modulation of UCN expression could influence important agronomical traits. This work describes hitherto unknown UPF1 associated factors, their study will provide novel insights into the mechanisms involved in the balance between mRNA translation, storage and decay in plants.

La ppGpp et la pppGpp, qui sont synthétisées/hydrolysées par les RelA/Spot homologs (RSH), jouent un rôle centrale dans l’adaptation des bactéries contre la privation des nutriments et les stress environnementaux. Les enzymes RSH et ppGpp ont été découverts dans le chloroplaste. Il a été récemment démontré que ppGpp joue un rôle comme répresseur globale de l’expression de gènes chloroplastiques. Certains stresses environnementaux et hormones induisent l’accumulation de ppGpp chez les plantes, cependant le mécanisme moléculaire n’est pas encore connu. Ici nous nous sommes intéressés à découvrir les facteurs qui interagissent avec les RSH, et qui donc sont susceptible de réguler le métabolisme du ppGpp. En utilisant un crible double-hybridation de levure nous avons identifiées des proteines qui interagissent avec les RSH y compris l’acyl carrier protein (ACP) et des GTPases associées au ribosome. ACP et RSH1 semblent être indispensables pour l’accumulation de ppGpp induite par la carence de la biosynthèse des acides gras, tandis que le ppGpp et un GTPase associé au ribosome contribuent à la résistance contre le heat-shock. Nous avons aussi effectué du co-immunoprécipitation spectrométrie de masse avec RSH1. Plusieurs protéines ont été identifiées y compris des protéines associées au nucléoid et des protéines liées à la signalisation chloroplastique, indiquant que RSH1 pourrait etre impliqué dans la machinerie de transcription chloroplastique. Nos résultats montrent que chez les plantes le ppGpp joue un rôle non seulement comme chez les bacteriés mais aussi participe à de nombreux processus biologiques qui sont spécifiques aux plantes. / Guanosine tetra-phosphate and penta-phosphate (ppGpp and pppGpp), which are synthesized/hydrolyzed by RelA/Spot homolog (RSH) enzymes, play a central role in the adaptation of bacteria to nutrient limitation or other stresses. Both RSH enzymes and ppGpp are present in the chloroplasts of plants. Recent studies have shown that ppGpp acts as a global repressor of chloroplast gene expression. Certain environmental stresses and hormones induce ppGpp accumulation in chloroplasts, however the molecular mechanisms underlying the activation of ppGpp signalling in response to such stimuli is essentially unknown. We searched for factors that interact with RSH enzymes and so could play a role in activating ppGpp signalling. Using a targeted yeast two hybrid screen several proteins were identified that interact with RSH enzymes including acyl carrier protein (ACP) and ribosome associated GTPases. ACP and RSH1 appear to be required for ppGpp induction in response to fatty acid biosynthesis depletion, while ppGpp and an RSH-interacting GTPase contribute to the resistance of plants to heat shock. We also performed non-targeted co-immunoprecipitation mass spectrometry (CoIP-MS) of RSH1. New RSH interaction candidates were identified, including plastid nucleoid associated proteins and chloroplast signalling proteins, suggesting that RSH1 may be associated with the plastid transcription machinery. Our results give new insights into ppGpp signalling, and show that some elements are conserved between plants and bacteria, while others are implicated in plant-specific biological processes.

Le travail présenté dans ce manuscrit a permis de définir un nouveau rôle de l’uridylation des ARNm en utilisant Arabidopsis comme organisme d’étude. L’uridylation des ARN est catalysée par des ARN nucléotidyltransférases de la famille des poly(A) polymérases non canoniques ou ncPAP. Parmi les 14 gènes codant pour des ncPAP chez Arabidopsis, nous avons identifié une terminale uridylyl transférase, TUT1, responsable de l’uridylation des ARNm. Nos résultats montrent que TUT1 uridyleles ARNm après une étape de déadénylation. Cette uridylation ne modifie pas la vitesse de dégradation des ARNm mais est essentielle pour prévenir l’attaque des extrémités 3’ des ARNm déadénylés par des activités 3’-5’ exoribonucléasiques et la formation de transcrits aberrants tronqués en 3’. De manière intéressante, cette protection par l’uridylation peut être détectée au niveau des polysomes. Une des fonctions biologiques de l’uridylation des ARNm consiste à établir une polarité de 5’ en 3’ de la dégradation des ARNm. Cette polarité pourrait être essentielle dans le cas d’une dégradation des ARNm en cours de traduction. / The work presented in this manuscript defines a new role of mRNA uridylation, using Arabidopsis as a model organism. RNA uridylation is catalyzed by RNA nucleotidyltransferases belonging to the non canonical poly(A) polymerase (ncPAP) family. Among the 14 genes encoding ncPAPs in Arabidopsis, we identified a terminal uridylyl transferase, TUT1, responsible for mRNA uridylation. Our results show that mRNAs are uridylated by TUT1 after a deadenylation step. Uridylation doesn’t modify mRNA degradation rates but is essential for deadenylated mRNA 3’ end protection against 3’- 5’ exoribonucleolytic attacks and to prevent 3’ truncated aberrant mRNA formation. Interestingly, this protection by uridylation is detected in polysomes. One biological function of mRNA uridylation is to establish a 5’-3’ mRNA degradation polarity that could be essential in the case of cotranslational mRNA decay.

La production de biocarburants de deuxième génération basée sur la transformation de la biomasse végétale est une question d’actualité. La biomasse végétale est représentée par les parois des cellules, qui consistent en un réseau de microfibrilles de cellulose et de polysaccharides enchâssés dans de la lignine. Pour exploiter pleinement le potentiel de cette biomasse, il est nécessaire d’apporter des connaissances complémentaires sur les mécanismes de biosynthèse de ces polymères pariétaux. Par exemple, il est important d’améliorer le rendement de saccharification des microfibrilles de cellulose afin de produire de plus grandes quantités de bioéthanol. Nous avons donc combiné des études basées sur le modèle bien connu Arabidopsis et sur Brachypodium distachyon, la nouvelle espèce modèle pour les graminées tempérées et les céréales monocotylédones dédiées à la production de biocarburants. La cellulose est synthétisée par des complexes membranaires de cellulose synthases (CSC) qui contiennent les sous-unités catalytiques de cellulose synthase (CESAs), et cela requiert d’autres partenaires parmi lesquels KOR1, une endo-β-1,4-glucanase. Le trafic intracellulaire des CESAs semble jouer un rôle crucial dans la régulation du niveau de la synthèse de la cellulose. Nous avons étudié en détails le trafic intracellulaire de KOR1 dans des hypocotyles d’Arabidopsis cultivés à l’obscurité. En parallèle, lors d’un crible visuel de la collection de mutants de Brachypodium de l’INRA de Versailles, nous avons sélectionné un mutant nommé spa. Ce mutant partage des caractéristiques avec les mutants brittle culm du riz et de l’orge, comme par exemple des tiges cassantes, un xylème irrégulier, et une importante déficience en cellulose, surtout au niveau des tiges, qui contiennent 50% de la quantité retrouvée dans une plante sauvage. Des dosages de lignine ont montré une augmentation significative chez spa. De façon itnéressante, ce mutant présente un défaut flagrant du port érigé, au contraire des mutants brittle culm qui sont parfaitement érigés. Les défauts mécaniques du mutant spa s’illustrent par un module de Young trois fois inférieur à celui d’une plante sauvage. Des approches complémentaires ont été mise en œuvre afin d’identifier les défauts génétiques responsables de ce phénotype : le séquençage de gènes candidats reliés à la synthèse de la cellulose a été réalisé ainsi qu’une approche de NGS. De plus, dans le cadre du projet Européen RENEWALL et du projet KBBE CellWall, et grâce à l’outil BradiNet (M.Mutvil, KBBE CellWall) permettant d’accéder aux réseaux de co-expression, des stratégies RNAi sont en cours afin d’inactiver certains gènes seléctionnés selon des critères d’expression spécifiques, et selon leur implication potentielle dans la synthèse de la cellulose, spécifiquement chez les monocotylédones. Parmi ces gènes nous nous sommes concentrés sur la famille des MAP65 (Microtubules Associated Proteins), qui pourraient, au vu de la relation étroite entre microtubules et microfibrilles, jouer un rôle dans la déposition de la cellulose. / The production of second-generation biofuels based on the transformation of plant biomass is a pressing issue. Biomass is represented by cell walls of the plant cells consisting of a network of cellulose microfibrils and polysaccharides encrusted by lignin. To enhance the potential of plant biomass, we need to provide insights on the mechanisms of the biosynthesis of cell wall polymers. For example, it is important to improve the saccharification yield of cellulose microfibrils to produce the highest amount of bioethanol. We therefore combine studies on the well-known model plant Arabidopsis and Brachypodium distachyon, the new model species for temperate graminae and monocotyledonous crops dedicated to biofuel production. Cellulose is synthesized by plasma membrane-bound cellulose synthase complexes (CSC) containing cellulose synthase proteins (CESAs) and requires other partners among which the endo-beta1,4 glucanase KOR1. The intracellular trafficking of CESAs seems to be crucial to regulate the cellulose synthesis rate. We investigated in detail the intracellular trafficking of KOR1 in Arabidospis dark-grown hypocotyls.In parallel we selected by visual screening of the Versailles collection of mutagenized Brachypodium distachyon a mutant called spa. This mutant shares characteristics of the brittle culm mutants of rice and barley, such as brittleness, irregular xylem, and a cellulose content deficiency especially in stems, with 50% of the amount found in the wild type. Lignin assays indicate a higher amount of lignin in spa. Interestingly, this mutant is also "floppy" unlike others brittle culm mutants which are fully erected and the mechanical strength defects of spa is illustrated by a Young’s modulus three times lower than that of WT. Complementary approaches were used to identify the SPA gene: sequencing of candidate genes related to cell wall synthesis or co-expressed with secondary cell wall cellulose synthases and a classical mapping strategy combined with NGS methods. Moreover within the framework of the European RENEWALL and KBBE CellWall projects and thanks to the co-expression network tool BradiNet (M. Mutwil, KBBE project), RNAi strategies are in progress to inactivate a few genes selected according to specific expression criteria and potentially involved in cell wall synthesis specifically in monocots. Among these genes we are focusing on the MAP65 family (Microtubules Associated Proteins), which could play a role in cellulose deposition according to the close relationship between microfibrils and microtubules.

Afin de mieux comprendre les mécanismes du trafic cellulaire, les processus de transport des substrats vacuolaires à travers le tonoplaste, le stockage des métabolites et leur dégradation, une analyse globale et exhaustive du protéome vacuolaire d'Arabidopsis thaliana a été réalisée. La connaissance de la localisation subcellulaire des protéines permet de mieux comprendre la fonction des organelles et la compartimentation du métabolisme des plantes. Mais la description précise du protéome d'un organite nécessite d'identifier clairement les véritables protéines résidantes du compartiment étudié. Une tâche si précise est complexe puisqu'elle nécessite la mise en place d'une préparation d'organites purs et homogènes. Pour y parvenir, un protocole de purification de vacuoles à partir de protoplastes isolés de cellules en culture sur un gradient de densité de Ficoll a été amélioré. La combinaison de plusieurs approches de protéomique a permis d'identifier les protéines présentes dans les fractions vacuolaires soluble et membranaire de façon quantitative et fonctionnelle. Les différentes approches ont ainsi mis en évidence des associations et mécanismes moléculaires complexes qui régissent les différentes activités vacuolaires. Cette protéothèque de référence constitue une base pour étudier la dynamique du protéome vacuolaire en réponse à plusieurs stress incluant les métaux lourds. Plusieurs méthodes sans a priori et ciblée ont été proposé afin d'étudier l'impact du cadmium sur la vacuole, ce compartiment cellulaire clé de la détoxication. / To better understand the mechanisms governing cellular traffic, transport process of substrates across the tonoplast, storage of various metabolites and their ultimate degradation, a comprehensive and thorough analysis of Arabidopsis thaliana vacuolar proteome was performed. Protein subcellular localization knowledge is an important step toward assigning functions of organelles and plant metabolism compartmentation. But confident description of proteome organelle content requires clear identification of the true resident proteins of the studied compartment. This task involves pitfalls and requires that either organelle preparations are free of contaminants or that techniques are used to discriminate between genuine organelle residents and contaminating proteins. To achieve this, vacuoles purification protocol from protoplasts on a Ficoll density gradient has been improved. The combination of several proteomic approaches attempt to present soluble and membrane vacuolar proteins in a quantitative and functional manner. Different approaches have thus shown associations and complex molecular mechanisms that govern the various vacuolar activities. The constitute proteins library provides references to study the vacuolar proteome dynamics in response to different stresses including heavy metals. Many methods without a priori or targeted were proposed to study the impact of cadmium on the vacuole, the key cell compartment of detoxification. Proteomics provides powerful tools for characterizing the protein contents of vacuoles during cadmium stress.

La protéine « Selenium Binding Protein » (SBP1), présente chez la plupart des êtres vivants, a un rôle qui n'est pas encore élucidé. Cette protéine possède dans sa structure primaire de nombreux sites potentiels de liaisons aux métaux. Chez Arabidopsis thaliana, la surexpression de SBP1 augmente la tolérance à deux composés toxiques pour la plante, le cadmium (Cd) et le sélénium (Se) qui sont présents dans les sols pollués. Pour mieux comprendre la fonction de SBP1 dans les mécanismes de détoxication, une démarche intégrée combinant deux approches complémentaires, in vitro et in planta, a été menée. La caractérisation biochimique de SBP1 a mis en évidence ses propriétés chélatrices vis-à-vis de différents ligands dont le Cd et le Se. Le Cd se lie à SBP1 avec un ratio molaire ligand/SBP1 de 3 et un KD de 2,2 × 10-7 M via principalement des acides aminés soufrés et potentiellement à moindre échelle des résidus histidines. Le Se, initialement sous forme SeO32-, se lie à SBP1 de façon covalente, avec un ratio molaire ligand/SBP1 de 1, via les cystéines 21 et 22 pour former une liaison de type R-S-Se-S-R. Les analyses in planta de localisation subcellulaire ont montré que SBP1 était à la fois cytosolique et nucléaire. L'utilisation de lignées bioluminescentes a permis d'analyser la zone promotrice du gène SBP1. Le motif cis ‘GAGAC' connu pour être impliqué dans la régulation de certains gènes à une carence en soufre (S) a été identifié comme élément cis majeur de la régulation de l'expression de SBP1 en réponse à différents stress, dont le Cd et le Se. Ce résultat démontre l'existence d'un lien entre la fonction de SBP1 et une demande en S de la cellule. La surexpression de SBP1 in planta perturbe le niveau d'accumulation du Se dans les parties aériennes, mais pas sa spéciation. L'ensemble de ces résultats semble indiquer que SBP1 soit impliquée dans des mécanismes de détoxication via ses propriétés chélatrices et qu'elle joue également un rôle dans le métabolisme du S et du Se. / The function of the protein “Selenium binding protein 1” (SBPP1), present in almost all organisms, is not yet well established. This protein has numerous potential metal binding sites. In Arabidopsis thaliana, SBP1 overexpression increases tolerance to two toxic compounds for the plant, cadmium (Cd) and selenium (Se), which are often found as soil pollutants. For a better understanding of SBP1 function and its involvement in detoxification mechanisms, an integrated approach combining in vitro and in planta experiments, has been performed. Biochemical characterization of SBP1 has revealed its chelating properties to different ligands including Cd and Se. Cd is bound to SBP1 with a metal ion to protein molar ratio of 3 and a KD of 2.2 × 10-7 M mainly via sulfur-containing amino acids and potentially histidine residues. Se from SeO32- can covalently bound SBP1 with a ligand to protein molar ratio of 1. This binding occurs via cysteines 21 and 22 and forms a R-S-Se-S-R complex. In planta analyses have shown that SBP1 is cytosolic and nuclear. The use of bioluminescent lines allowed the identification of a GAGAC motif in the SBP1 promoter region. This motif is a sulfur starvation responsive element and a major cis element involved in SBP1 expression in response to stress, including Cd and Se. The result directly links SBP1 function to an enhanced sulfur demand of the cell. SBP1 over expression in plants disturbs Se accumulation in shoots but not its speciation. All together these results strongly suggested that SBP1 could act as a detoxifying protein through its chelating properties and plays a role in S/Se metabolisms.

Orientadores: Ione Salgado, Cecilia Alzira Pinto-Maglio / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T11:22:55Z (GMT). No. of bitstreams: 1 Seligman_Kelly_M.pdf: 3879828 bytes, checksum: 479952026f21e4da513fff98938d5538 (MD5) Previous issue date: 2008 / Resumo: O radical óxido nítrico (NO), que pode ser produzido nos organismos pela oxidação de arginina ou redução de nitrito, é uma importante molécula sinalizadora em plantas atuando como modulador de diversos processos metabólicos e de desenvolvimento. Recentemente foi identificado como um dos sinais envolvidos no processo de floração. A transição da fase de crescimento vegetativo para a fase reprodutiva é atrasada em plantas mutantes que superproduzem NO, enquanto que a floração é precoce em plantas mutantes deficientes na síntese deste radical. O principal objetivo deste trabalho foi identificar os sítios de produção de NO durante o desenvolvimento floral de Arabidopsis thaliana L. Foram utilizados os indicadores fluorescentes 4,5-diacetato de diaminofluoresceína (DAF-2 DA) e 1,2-diaminoantraquinona (1,2-DAA) para localizar in situ, por microscopia de fluorescência, a produção de NO em botões florais de A. thaliana em diferentes estágios de desenvolvimento. Ainda, a produção de NO pelas estruturas florais foi comparada entre plantas do tipo selvagem e mutante duplo defectivo para os genes estruturais da enzima nitrato redutase - NR - (nia1 nia2) que apresentam conteúdo reduzido de aminoácidos e nitrito e, consequentemente, de NO em suas folhas. Foi analisado também o efeito do seqüestrador de NO, CPTIO, na prevenção da emissão de fluorescência. Os resultados mostraram que o NO é sintetizado em células e tecidos específicos da estrutura floral e que sua produção aumenta com o desenvolvimento floral até a antese: a fluorescência dos indicadores, prevenida pelo seqüestrador de NO, ficou restrita às papilas estigmáticas em gineceus imaturos e a grãos de pólen produzidos pela antera no estame. Plantas mutantes de A. thaliana nia1 nia2 apresentaram o mesmo padrão de emissão de NO nos órgãos florais que o tipo selvagem. Sépalas e pétalas não apresentaram produção significativa de NO em ambos os genótipos analisados. A validação dos resultados obtidos por microscopia foi feita através da incubação de tecidos florais intactos com DAF-2, quantificando-se, por espectrofluorimetria, o composto DAF-2T resultante da reação do indicador com o NO emitido pelos tecidos. Os dados obtidos por espectrofluorimetria mostraram que a intensidade de fluorescência emitida por botões florais foi maior durante o estágio 11 de desenvolvimento, corroborando os dados obtidos de localização por microscopia de fluorescência. Ainda, foi possível quantificar que as plantas do tipo selvagem apresentam, em média, maior intensidade de fluorescência emitida, que plantas nia1 nia2: 1,39 e 1,89 vezes maior nas fases 11 e 13 de desenvolvimento, respectivamente. Um segundo objetivo deste trabalho foi avaliar o papel da enzima NR na indução floral. Os dados obtidos revelam que plantas nia1 nia2 possuem floração precoce, 6 dias em média, quando comparadas com plantas do tipo selvagem. Para verificar se a floração precoce no mutante nia1 nia2 era conseqüência apenas da deficiência do radical NO, ou também da deficiência de aminoácidos, foram analisados os parâmetros de indução floral em plantas nia1 nia2 tratadas com os aminoácidos arginina ou glutamina, para a recuperação dos níveis basais de aminoácidos. A floração neste mutante permaneceu precoce, sugerindo que o fenótipo de floração precoce é conseqüência da deficiência de NO nestas plantas durante a fase vegetativa. Estes resultados sugerem que o NO pode ter um importante papel no processo de floração e no sucesso da reprodução vegetal / Abstract: The radical nitric oxide (NO), that can be produced in the organism by arginine oxidation or nitrite reduction, is an important signaling molecule in plants acting as modulator of several metabolic and developmental processes. Recently NO was identified as one of several signals involved in flowering. The transition from vegetative to reproductive growth is delayed in mutant plants that overproduce NO, while this process is precocious in mutant plants deficient in NO synthesis. The main objective of this study was to identify the sites of NO production during floral development of Arabidopsis thaliana. The fluorescent probes, 4,5-daminofluorescein diacetate and 1,2-diaminoanthraquinone, were utilized to localize in situ, by fluorescence microsopy, the NO production in floral buds of A. thaliana at different stages of development. Still, NO production by the floral structures was compared between wild-type and double defective mutant plants for structural genes of nitrate reductase enzyme - NR - (nia1 nia2) which have reduced content of amino acids and nitrite and, consequently, of NO in their leaves. It was also analyzed the effect of the NO scavenger, CPTIO, in preventing the fluorescence emission. The results showed that NO is synthesized in specific cells and tissues in the floral structure and its production increases with the floral development until anthesis: the fluorescence of the indicators, prevented by NO scavenger, was restricted to the stigmatic papillae, in the gynoecium, and to pollen grains produced by anther in stamen. Mutant plants of A. thaliana nia1 nia2 showed the same pattern of NO emission by the floral organs to that observed in the wild type genotype. Sepals and petals showed no significant NO production in both genotypes analyzed. Validation of the results obtained by fluorescence microscopy was realized by incubating floral tissues with DAF-2 and quantifying, by spectrofluorimetry, the DAF-2T resulted from reaction between the probe and the NO emitted by the tissues. Data obtained by spectrofluorimetry showed that the fluorescence intensity emitted by floral buds was higher during stage 11 of development, corroborating localization data obtained by fluorescence microscopy. Additionally, wild type plants showed greater intensity of fluorescence emission, on average, than nia1 nia2 plants: 1,39 and 1,89 times higher in phases 11 and 13 of development, respectively. A second objective of this work was to evaluate the role of the NR enzyme in floral induction. Data obtained revealed that nia1 nia2 plants flowers earlier, 6 days on average, than the wild-type plants. To verify whether the early flowering phenotype in nia1 nia2 was only consequence of the NO deficiency, or was also due to the amino acids deficiency, it were analyzed the parameters of floral induction in nia1 nia2 plants treated with the amino acids arginine or glutamine for the recovery of the amino acids levels. Flowering in this mutant remained precocious, suggesting that the early flowering phenotype is a consequence of NO deficiency in these plants during the vegetative growth. These results suggest that NO may have an important role in the flowering process and to the reproductive success of the plant / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Submitted by MARCOS LEANDRO TEIXEIRA DE OLIVEIRA (marcosteixeira@ufv.br) on 2018-09-06T11:28:25Z No. of bitstreams: 1 texto completo.pdf: 2396350 bytes, checksum: d4a4b3c74df2af6f3609cee963d570e2 (MD5) / Made available in DSpace on 2018-09-06T11:28:25Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2396350 bytes, checksum: d4a4b3c74df2af6f3609cee963d570e2 (MD5) Previous issue date: 2018-07-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A via de sinalização de morte celular mediada por proteínas NRP/DCDs foi primeiramente descrita em soja como uma via integrativa de resposta a múltiplos estresses. A falta de ferramentas moleculares adequadas à caracterização dessa via em soja levou ao estudo de sua conservação em espécies modelo. Em Arabidopsis, os membros da via foram descritos, no entanto não foi definido o ortólogo do gene GmNAC30. Neste trabalho, foi conduzida uma análise in silico da subfamília SNAC-A (ATAF), à qual pertence GmNAC30, demonstrando ser ela amplamente distribuída no reino vegetal. Em comparação com Arabidopsis, foi verificada uma expansão dessa subfamília em soja, o que pode estar relacionado à pressão seletiva que espécies cultiváveis sofrem a favor da manutenção de genes capazes de conferir tolerância a estresses. Consistente com essa ideia, a análise da expressão de alguns membros desse subgrupo em soja demonstrou que todos eles respondem a múltiplos estresses. Os genes duplicados da subfamília SNAC-A em soja exibiram padrão e cinética de indução por polietilenoglicol (PEG) e Tunicamicina (TUN) similares; porém, os pares apresentaram diferenças sutis de indução por AS, indicando que devem possuir funções parcialmente sobrepostas, mas não absolutamente idênticas. A fim de verificar a hipótese difundida de ser ATAF1 o ortólogo de GmNAC30 foram realizados ensaios de interação de ATAF1 com ANAC036 (ortólogo de GmNAC081) pelo sistema de duplo-híbrido de leveduras e análises de expressão gênica de ATAF1 em resposta a estresses osmótico e no retículo endoplasmático (RE). Os resultados obtidos, no entanto, indicaram não ser ATAF1 o ortólogo de GmNAC30, uma vez que não interagiu com ANAC036 pelo sistema de duplo- híbrido, e ao contrário de GmNAC030, não foi induzido por tunicamicina (TUN), embora, assim como GmNAC030, ATAF1 tenha exibido atividade transcricional pelo sistema de mono-híbrido de leveduras. Uma possível interação do gene ATAF1 com a via de morte celular induzida por estresses no RE e osmótico foi avaliada por genética reversa no mutante ataf1-2 que exibiu 75% de redução na expressão de ATAF1. Tanto a expressão basal quanto a expressão induzida por PEG e TUN dos genes NRP-1 e ANAC036, componentes da via de morte celular, foi superior no mutante ataf1-2 do que em Col-0, implicando ATAF1 como possível regulador negativo da via de morte celular mediada por NRPs. Sendo ATAF2 um outro membro do subgrupo SNAC-A que está filogeneticamente muito próximo a GmNAC30, foi avaliada a sua resposta a tratamentos com PEG e tunicamicina em plântulas das linhagens Col-0 e ataf1-2. Foi observada a indução de ATAF2 em resposta a ambos os estresses, sendo que a indução é aumentada no mutante ataf1-2, consistente com o perfil de expressão gênica de integrantes da via de morte celular mediada por NRPs. Coletivamente, estes resultados substanciam o argumento de que ATAF2 possa ser ortólogo de GmNAC030. No entanto, experimentos complementares são necessários para a confirmação dessa hipótese. / The NRP/DCDs- mediated cell death signaling was first described in soybean as an integrative pathway for multiple stresses. The lack of adequate molecular tools for characterization of this pathway in soybean led to the demonstration of its conservation in plant model systems. In Arabidopsis, several members of this family have been described, but the GmNAC30 ortholog remains to be identified. In this investigation, an in silico analysis of the SNAC-A (ATAF) subfamily of GmNAC30 was conducted and demonstrated that SNAC-A is largely distributed in the plant kingdom. As compared to Arabidopsis, the SNAC-A subfamily was expanded in soybean, more likely due to the selective pressure undergone by economically relevant crops towards the maintenance and duplication of stress related genes. Accordingly, an expression analysis of some soybean genes of this subfamily demonstrated that they respond to multiple stresses. The duplicated genes from the soybean SNAC-A subfamily displayed similar profile and induction kinetics by PEG and tunicamycin (TUN). Nevertheless, the pairs exhibited slightly differences in salicylic acid (SA) induction, indicating that they may display partially overlapping, but not absolutely identical functions. To confirm that ATAF1 is a GmNAC030 ortholog, yeast two-hybrid assays for ATAF1-ANAC036 (GmNAC081 otrholog) interaction and analysis of ATAF1 gene expression in response to osmotic and endoplasmic reticulum (ER) stresses were conducted. However, the results indicated that ATAF1 is not likely a GmNAC30 ortholog, as ATAF1 did not interact with ANAC036 in yeast and, opposing to the GmNAC030 expression profile, it was not induced by TUN, although like GmNAC030, ATAF1 was transcriptionally active by the yeast mono-hybrid assay. A possible interaction of ATAF1 with the ER stress- and osmotic stress-induced cell death signaling was monitored by reverse genetic in ataf1-2 mutant, which displayed a 75% reduction in the accumulation of ATAF1 transcripts. Both basal and stress-induced expressions of NRP-1 and ANAC036, components of the cell death pathway, were higher in ataf1-2 than in Col-0, implicating ATAF1 as a possible negative regulator of the NRPs- mediated cell death signaling. Because ATAF2 is another member of the SNAC-A subfamily, which is phylogenetically close related to GmNAC30, its response to PEG and TUN was monitored in Col-0 and ataf1-2 seedlings. ATAF2 was induced by both treatments and the transcript accumulation was further increased in ataf1-2, which was consistent with the expression profile of components of the NRPs-mediated cell death signaling. Collectively, these results substantiate the argument that ATAF2 may be a GmNAC030 ortholog. However, complementary experiments are necessary to confirm this hypothesis.