La formation des fruits charnus est un processus de développement impliquant trois stades principaux : (i) la transition fleur/fruit ou nouaison, (ii) la croissance et enfin (iii) la maturation des fruits. Chacune de ces étapes correspond à une transition développementale associée à d’importants changements physiologiques et structurels. Parmi toutes les hormones, l’auxine est connue pour jouer un rôle important dans l‟initiation et la coordination du processus de nouaison et des phases précoces de développement du fruit. La mise en place de la réponse à l‟auxine nécessite l‟intervention de facteurs de transcription appartenant à la famille des ARF (Axin Response Factor) connus pour réguler l‟expression des gènes de réponse précoce à l‟hormone en se liant aux Cis-éléments de type AuxRE (Auxin Response Element) possédant le motif conservé de réponse à l‟auxine. Les ARF sont de ce fait des candidats forts pour faire partie du mécanisme moléculaire par lequel l’auxine intervient dans le processus de nouaison. Le projet de recherche réalisé au cours de la thèse a permis d‟isoler et de caractériser au total 22 gènes Sl-ARF chez la tomate (Solanum lycopersicum), la plante modèle pour l’étude du développement et de la maturation des fruits charnus. Les gènes Sl-ARF montrent des profils d‟expression distincts selon les tissus et organes considérés, suggérant des fonctions spécifiques pour les membres de cette famille multigénique. Il est de plus montré que certains gènes Sl-ARF sont régulés à la fois par l’auxine et par l’éthylène, suggérant qu‟ils participent potentiellement au dialogue entre les voies de signalisation des deux hormones. L’expression transitoire a révélé la capacité des Sl-ARF à agir comme activateur ou répresseur transcriptionnel des gènes de réponse à l’auxine. L‟étude des profils d’expression globale, réalisée par RNA-seq à l‟échelle du génome entier, a révélé pour la première fois l‟existence d‟un niveau important de régulation par épissage alternatif des ARFs pendant la transition fleur-fruit. La localisation nucléaire des protéines Sl-ARF8A / B a été déterminée par fusion avec le gène rapporteur GFP puis expression dans un système "signle cell". L‟étude d’expression a révélé des profils distinctifs entre ARF8A et ARF8B avec une augmentation notable des transcrits Sl-ARF8A suite à la pollinisation des fleurs. Le rôle physiologique du gène Sl-ARF8A a été par la suite abordé par une approche de génétique inverse fournissant un nouvel éclairage sur les événements moléculaires qui sous-tendent la mise à fruit. La surexpression de Sl- ARF8 dans la tomate engendre des phénotypes pléiotropiques touchant la croissance - 4 - végétative (réduction de la taille des plantes, altération du développement racinaire et des tiges latérales) et l‟appareil reproducteur avec la formation de fruits parthénocarpiques (absence de graines). L’analyse histologique a révélé une modification notable du placenta et des ovules chez les lignées de sur-expression de Sl-ARF8 et les études par RNA-Seq ont identifié plus de 2632 gènes différentiellement exprimés chez les surexpresseurs par comparaison avec les lignées non transformées. Au total, l‟étude réalisée au cours de la thèse fournit une description exhaustive de la famille des ARF chez la tomate et une caractérisation fonctionnelle du gène Sl-ARF8 qui souligne son rôle comme figure centrale du mécanisme de contrôle de la nouaison des fruits. / The making of a fleshy fruit is a developmental process involving three main stages known as (i) fruit set, (ii) fruit growth and (ii) fruit ripening each corresponding to a transition step associated with major physiological and structural changes. Among other hormones, auxin is known to play a dynamic role in triggering and coordinating the changes associated with the process of fruit set and early fruit development. Auxin responses are mediated at the transcriptional level by Auxin Response Factors (ARFs) which regulate early auxin-responsive genes by specific binding to TGTCTC Auxin Response Elements (AuxREs). ARFs are therefore good candidates for being among the components of the molecular mechanism by which auxin mediates the fruit set. In the present study, a total of 22 Sl-ARF genes have been isolated and characterized in tomato (Solanum lycopersicum), a model plant for the study of fleshy fruit development and ripening. Expression profiling revealed distinctive patterns for Sl-ARF genes in different tomato tissues. Hormone treatment indicated that Sl-ARFs can be regulated both by auxin and ethylene with Sl-ARF2B, 5 and 9 likely to be involved in the cross-talk between the two hormones. Transient expression using a single cell system uncovered the ability of Sl- ARFs to act either as transcriptional activator or repressor in regulating the expression of auxin-responsive genes. Genome-wide expression profiling performed by deep RNASequencing revealed for the first time the importance of the alternative splicing mode of regulation of ARF genes during tomato fruit set. The physiological significance of two closely related Sl-ARFs, Sl-ARF8A and Sl-ARF8B, was addressed in the present study via a reverse genetics approach providing new insight on the molecular events underlying tomato fruit set. Fusion to GFP reporter gene indicated that both Sl-ARF8A/B proteins are nuclear localized. Expression analysis by RT-qPCR revealed some distinctive features between Sl-ARF8A and Sl-ARF8B with a notable increase in Sl-ARF8A transcript upon flower pollination. Over-expression of Sl-ARF8A/B in tomato resulted in pleiotropic phenotypes, including dwarf plants, altered root and lateral shoot development and parthenocarpic fruits (seedless). Histological analysis revealed altered placenta and ovules development in SlARF8A-OX flowers and RNA-Seq profiling identified over 2632 differentially expressed (DE) genes in SlARF8A-OX flower buds compared to wild type control plants. Considering the dramatic change in gene expression of genes related to auxin, jasmonate and ethylene displayed in SlARF8A-OX lines, these phytohormones are likely to play an active role in coordinating the fruit set process. Altogether, the present - 6 - study provided a comphensive description of the tomato ARF gene family and a functional characterization of Sl-ARF8 defining this ARF member as a central figure of the control mechanism of the fruit set process.

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No. of bitstreams: 2 Dissertação - Carlos de Melos e Silva Neto - 2013.pdf: 671422 bytes, checksum: 7774a52856834c1b8270fa6218029c99 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-09-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Pollinators provide an essential service to the ecosystem and bring numerous benefits to society, through its role in the production of food in agriculture and the conservation of biological diversity. In tomato, anthers are poricidal, thus the release of pollen grains requires the presence of pollinators that vibrate these anthers. Thus, pollinators of tomatoes are actually bees that perform buzz pollination. Here, we observed aspects of floral biology, quantity and viability of pollen grains and ovules, and pollinators behavior and richness. To evaluate the effects of pollination in the amount of pollen on the stigmas, we selected 37 plants and bagged an inflorescence and let another available to pollinators. Each marked inflorescence had three stigmas removed the pollen grains on their surfaces were quantified. For comparison of fruit production, fruit development were followed in inflorescences bagged and not bagged for 40 days, and then the fruits were counted, weighed, measured, and had their seeds counted. The anthesis is at 6:30 am. The flower remains open until 6:00 pm, closing and reopening in the next day at the same time, lasting up to 73 hours. The greatest amount of pollen in the anther is in the early hours of the flower opening, with reduction and stabilization of the number of pollen grains around noon, coinciding with increased pollinator visitation. We found 25 species of bees in pollinating tomato crops. The abundance of bees coinciding with hours of availability and depletion of pollen suggests efficient removal of pollen from the anthers by the native bees The amount of pollen on the stigma of flowers available to pollinators was higher than on the stigma of bagged flowers. Fruit production was higher in open inflorescences than in bagged inflorescences. Native bees pollinate tomato flowers, increasing the pollen load on the stigma and fruit production. / Os polinizadores fornecem um serviço essencial ao ecossistema e trazem inúmeros benefícios à sociedade, através do seu papel na produção de alimento na agricultura e na conservação da diversidade biológica. No tomateiro, as anteras têm abertura poricida, assim, para liberação dos grãos de pólen é necessária a presença de polinizadores efetivos que realizam a vibração dessas anteras. Sendo assim, os polinizadores de fato dos tomateiros são abelhas que realizam polinização vibrátil. Na realização deste trabalho, foram observados aspectos da biologia floral como duração da flor, a quantidade e viabilidade de grãos de pólen e óvulos, além dos aspectos ecológicos dos polinizadores, como observação e coleta dos visitantes florais. Para avaliar os efeitos da polinização na quantidade de pólen do estigma, selecionamos 37 plantas, ensacamos uma inflorescência e deixamos outra disponível aos polinizadores. De cada inflorescência marcada, retiramos três estigmas e quantificamos os grãos de pólen em suas superfícies. Para a comparação da produção de frutos, acompanhamos o desenvolvimento dos frutos nas inflorescências ensacadas e não ensacadas durante 40 dias, e posteriormente os frutos foram contados, pesados, medidos e tiveram suas sementes contadas. A antese floral inicia às 6h30 da manhã. A flor permanece aberta até às 18h, fechando e no dia seguinte reabrindo no mesmo horário, durando até 73h. A maior oferta de pólen é nas primeiras horas da abertura da flor, com redução e estabilização do número de grãos de pólen por volta do meio dia, coincidindo com o aumento da visitação dos polinizadores. Foram encontradas 25 espécies de abelhas polinizadoras nos cultivos de tomateiro. A abundância das abelhas coincidindo com os horários de disponibilidade e esgotamento do pólen sugere eficiência na retirada dos grãos de pólen das anteras, muitos dos quais se aderem provavelmente ao estigma da própria flor, ocorrendo o posterior desenvolvimento dos frutos. A quantidade de grãos de pólen no estigma das flores disponíveis para os polinizadores foi maior do que no estigma das flores ensacadas. A produção de frutos foi maior em inflorescências não ensacadas que em inflorescências ensacadas. Os frutos não ensacados apresentaram mais sementes que os ensacados. As abelhas nativas polinizam as flores do tomateiro, aumentando a carga de pólen no estigma e a produção de frutos.