A genética molecular permite o entendimento dos mecanismos que regulam o desenvolvimento dos órgãos vegetais em resposta a fatores bióticos e abióticos. A regulação de vias gênicas é de fundamental importância no sucesso reprodutivo, evolutivo e econômico dos vegetais. Uma das modalidades de regulação é a pós transcricional através de microRNAs (miRNAs). Tratam-se de pequenos RNAs endógenos não codantes que possuem uma complementaridade quase perfeita em plantas. Em plantas, muitos genes-alvos de miRNA codificam-se para fatores de transcrição, como os genes SQUAMOSA Promoter-Binding Protein-Like (SPL/SBP). O microRNA156, conservado entre as Angiospermas, regula diversos fatores de transcrição da família SBP. O módulo miR156/SBP, denominado via da idade (AGE), atua ao longo do ciclo de vida dos vegetais regulando as transições de fase: juvenil-adulta e vegetativa-reprodutiva. O tomateiro, Solanum lycopersicum L., possui genes SlSBPs regulados pelo miR156 e com funcionalidade não esclarecida. Dentre aqueles, o gene SlSBP6c (Solyc12g038520) se destaca por possuir maior similaridade filogenética com SPL6s/SBP6s de solanáceas do que seus genes homólogos SlSBP6a e SlSBP6b o que sugere a hipótese de um possível ganho de função. Com o intuito de testar essa hipótese, o presente trabalho objetivou caracterizar funcionalmente o gene SlSBP6c. Para tanto, utilizou-se o genótipo 35S::rSlSBP6c, planta transformada de tomateiro cultivar Micro-Tom (MT) que super-expressa a versão resistente ao miR156 do gene SlSBP6, fusionada ao promotor viral 35S. Este material vegetal foi gerado pelo laboratório de Genética Molecular do Desenvolvimento Vegetal e cedido para execução desta pesquisa. O trabalho se desenvolveu em duas etapas: caracterização molecular e morfo-fisiológica. Primeiramente, os genes SlSBP6a, SlSBP6b e SlSBP6c de tomateiro foram analizados quanto a sua expressão ao longo do desenvolvimento em folhas e inflorescência. E, posteriormente, foram analisados a complexidade foliar, o tempo de florescimento, a transição do meristema vegetativo para o reprodutivo, o teor relativo de clorofila e a fotossíntese líquida. Os resultados obtidos demonstram que os genes SlSBP6a e SlSBP6c são semelhantes no padrão de expressão gênica na homologia das protéinas que o codificam, indicando similaridade funcional. A de-regulação do gene SlSBP6c leva ao aumento a complexidade foliar, o teor relativo de clorofila e a fotossíntese líquida. O atraso na transição do meristema vegetativo para o reprodutivo se evidencia por um florescimento tardio nas plantas que super-expressam o gene SlSBP6c. Os resultados demonstram que o gene SlSBP6c exerce funcionalidade no tomateiro atuando na transição de fase vegetativo-reprodutivo. / Molecular genetics allows the understanding of the mechanisms that regulate the development of plant organs in response to biotic and abiotic factors. The regulation of gene pathways is of fundamental importance in the reproductive, evolutionary and economic success of the plants. Research has been increasing the knowledge about post-transcriptional regulation through microRNAs (miRNAs). The miRNAs are small non-coding endogenous RNAs that have almost perfect complementarity in plants. Many miRNA target genes in plants are encoded by transcription factors, such as the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL / SBP) genes. MicroRNA156 is conserved among Angiosperms and regulates several transcription factors of the SBP family. The miR156 / SBP module, called the age pathway (AGE), acts throughout the life cycle of plants regulating phase transitions juvenile-adult and vegetative-reproductive. The tomato, Solanum lycopersicum L., has newly described and unintelligently regulated miR156 regulated SlSBPs. Among these, the gene SlSBP6c (Solyc12g038520) stands out for having greater phylogenetic similarity with solanaceous SBP6s than its homologous genes SlSBP6a and SlSBP6b, indicating a possible gain of function related to characteristics of this family of plants as fleshy fruits and composite leaves. In order to test this hypothesis the work aimed to characterize the SlSBP6c gene functionally. Using as a tool the 35S::rSlSBP6c genotype, transformed tomato plant micro-Tom (MT) that over-expresses the miR156 resistant version of the SlSBP6 gene, fused to the 35S viral promoter. The Laboratory of Molecular Genetics of Plant Development generated this plant material. The work was developed in two stages: molecular and morpho-physiological characterization. In the first the genes SlSBP6a, SlSBP6b and SlSBP6c of tomato were analyzed for their expression throughout the development in leaves and inflorescence. In the second, the leaf complexity, the flowering time, the transition from vegetative to the reproductive meristem, the relative chlorophyll content and the net photosynthesis were evaluated. The results obtained demonstrate that the SlSBP6a and SlSBP6c genes are very similar in the pattern of gene expression in the homology of the coding proteins, indicating a functional similarity. The SlSBP6c gene acts to increase leaf complexity, relative chlorophyll content and liquid photosynthesis. A late flowering in plants that overexpress the SlSBP6c gene evidences the delay in the transition from the vegetative to the reproductive meristem. The data set demonstrates that the SlSBP6c gene has important functionality in tomatoes acting on vegetative-reproductive phase transition.

A genética molecular permite o entendimento dos mecanismos que regulam o desenvolvimento dos órgãos vegetais em resposta a fatores bióticos e abióticos. A regulação de vias gênicas é de fundamental importância no sucesso reprodutivo, evolutivo e econômico dos vegetais. Uma das modalidades de regulação é a pós transcricional através de microRNAs (miRNAs). Tratam-se de pequenos RNAs endógenos não codantes que possuem uma complementaridade quase perfeita em plantas. Em plantas, muitos genes-alvos de miRNA codificam-se para fatores de transcrição, como os genes SQUAMOSA Promoter-Binding Protein-Like (SPL/SBP). O microRNA156, conservado entre as Angiospermas, regula diversos fatores de transcrição da família SBP. O módulo miR156/SBP, denominado via da idade (AGE), atua ao longo do ciclo de vida dos vegetais regulando as transições de fase: juvenil-adulta e vegetativa-reprodutiva. O tomateiro, Solanum lycopersicum L., possui genes SlSBPs regulados pelo miR156 e com funcionalidade não esclarecida. Dentre aqueles, o gene SlSBP6c (Solyc12g038520) se destaca por possuir maior similaridade filogenética com SPL6s/SBP6s de solanáceas do que seus genes homólogos SlSBP6a e SlSBP6b o que sugere a hipótese de um possível ganho de função. Com o intuito de testar essa hipótese, o presente trabalho objetivou caracterizar funcionalmente o gene SlSBP6c. Para tanto, utilizou-se o genótipo 35S::rSlSBP6c, planta transformada de tomateiro cultivar Micro-Tom (MT) que super-expressa a versão resistente ao miR156 do gene SlSBP6, fusionada ao promotor viral 35S. Este material vegetal foi gerado pelo laboratório de Genética Molecular do Desenvolvimento Vegetal e cedido para execução desta pesquisa. O trabalho se desenvolveu em duas etapas: caracterização molecular e morfo-fisiológica. Primeiramente, os genes SlSBP6a, SlSBP6b e SlSBP6c de tomateiro foram analizados quanto a sua expressão ao longo do desenvolvimento em folhas e inflorescência. E, posteriormente, foram analisados a complexidade foliar, o tempo de florescimento, a transição do meristema vegetativo para o reprodutivo, o teor relativo de clorofila e a fotossíntese líquida. Os resultados obtidos demonstram que os genes SlSBP6a e SlSBP6c são semelhantes no padrão de expressão gênica na homologia das protéinas que o codificam, indicando similaridade funcional. A de-regulação do gene SlSBP6c leva ao aumento a complexidade foliar, o teor relativo de clorofila e a fotossíntese líquida. O atraso na transição do meristema vegetativo para o reprodutivo se evidencia por um florescimento tardio nas plantas que super-expressam o gene SlSBP6c. Os resultados demonstram que o gene SlSBP6c exerce funcionalidade no tomateiro atuando na transição de fase vegetativo-reprodutivo. / Molecular genetics allows the understanding of the mechanisms that regulate the development of plant organs in response to biotic and abiotic factors. The regulation of gene pathways is of fundamental importance in the reproductive, evolutionary and economic success of the plants. Research has been increasing the knowledge about post-transcriptional regulation through microRNAs (miRNAs). The miRNAs are small non-coding endogenous RNAs that have almost perfect complementarity in plants. Many miRNA target genes in plants are encoded by transcription factors, such as the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL / SBP) genes. MicroRNA156 is conserved among Angiosperms and regulates several transcription factors of the SBP family. The miR156 / SBP module, called the age pathway (AGE), acts throughout the life cycle of plants regulating phase transitions juvenile-adult and vegetative-reproductive. The tomato, Solanum lycopersicum L., has newly described and unintelligently regulated miR156 regulated SlSBPs. Among these, the gene SlSBP6c (Solyc12g038520) stands out for having greater phylogenetic similarity with solanaceous SBP6s than its homologous genes SlSBP6a and SlSBP6b, indicating a possible gain of function related to characteristics of this family of plants as fleshy fruits and composite leaves. In order to test this hypothesis the work aimed to characterize the SlSBP6c gene functionally. Using as a tool the 35S::rSlSBP6c genotype, transformed tomato plant micro-Tom (MT) that over-expresses the miR156 resistant version of the SlSBP6 gene, fused to the 35S viral promoter. The Laboratory of Molecular Genetics of Plant Development generated this plant material. The work was developed in two stages: molecular and morpho-physiological characterization. In the first the genes SlSBP6a, SlSBP6b and SlSBP6c of tomato were analyzed for their expression throughout the development in leaves and inflorescence. In the second, the leaf complexity, the flowering time, the transition from vegetative to the reproductive meristem, the relative chlorophyll content and the net photosynthesis were evaluated. The results obtained demonstrate that the SlSBP6a and SlSBP6c genes are very similar in the pattern of gene expression in the homology of the coding proteins, indicating a functional similarity. The SlSBP6c gene acts to increase leaf complexity, relative chlorophyll content and liquid photosynthesis. A late flowering in plants that overexpress the SlSBP6c gene evidences the delay in the transition from the vegetative to the reproductive meristem. The data set demonstrates that the SlSBP6c gene has important functionality in tomatoes acting on vegetative-reproductive phase transition.

Les plantes présentent une incroyable diversité de tailles, formes et couleurs, étroitement liée à certaines de leurs fonctions biologiques telles que la photosynthèse, la reproduction, etc. De ce fait, la façon dont ces organismes multicellulaires acquièrent des formes complexes est une question clé en biologie du développement. La morphologie des organes végétaux résulte en effet de la modulation, à l’échelle cellulaire, de patrons d’expression génétique, de croissance et de différenciation. Bien que la morphogénèse ait été largement étudiée d’un point de vue moléculaire, nous ne savons toujours pas comment ces réseaux génétiques sont traduits en formes biologiques. Le but de ce projet de recherche est donc d’étudier le développement des organes latéraux (feuilles juvéniles, feuilles caulinaires et organes floraux, id sépales, pétales et anthères) chez l’espèce modèle Arabidopsis thaliana. Afin d’approcher la question du rôle des interactions complexes entre cellules et organes lors du développement, nous nous intéressons à la variabilité entre les organes, mais aussi à la variabilité cellulaire intrinsèque de chaque organe. Nous avons donc testé (1) si la diversité de formes observées chez les organes latéraux résulte de modulations d’un programme développemental commun; (2) si la croissance et le développement des organes latéraux est un phénomène stochastique ou dépend de mécanismes sous-jacents spécifiques. Pour ce faire, nous utilisons une approche multidisciplinaire basée sur la génétique, la microscopie confocale et l’analyse d’images 3D pour extraire les patrons de croissance inhérents aux différents organes. Les résultats de la première étude (Chapitre 2) montrent que la forme des organes dépend de l’équilibre entre croissance et différentiation, dont la régulation précise permet l'acquisition de fonctions hautement spécialisées. La feuille caulinaire, par exemple, présente un retard de différenciation qui permet une activité morphogénétique prolongée et une redistribution de la croissance. À travers la suppression transitoire de la croissance lors des premiers stades de développement, la trajectoire développementale de la feuille caulinaire permet sa double fonction, à la fois protectrice et photosynthétique.\par La deuxième étude (Chapitre 3), quant-à-elle, s’intéresse aux comportements des cellules individuelles, dont la croissance, bien que contrôlée par des informations positionnelles, est souvent hétérogène. Cette variabilité résulte de la différenciation de cellules spécialisés, les stomates, qui suivent un programme de développement spécifique. Le comportement autonome de ces cellules, asynchrone, est la principale source de variabilité dans des tissus dont la croissance est autrement homogènes. Dans l’ensemble, cette thèse a permis de mettre en lumière l’importance de la temporalité lors du développement des organes végétaux. Que ce soit à l’échelle de l’organe, du tissu ou de la cellule, la modulation et la synchronisation de la croissance et de la différentiation sont nécessaires à l’acquisition des formes stéréotypiques des organes et à leur complexité fonctionnelle. / Plants display an incredible diversity of sizes, shapes, and colors, closely linked to some of their biological functions, such as photosynthesis, reproduction, etc. How these multicellular organisms acquire complex shapes is, therefore, a key question in developmental biology. The morphology of plant organs results from cell-level modulation of patterns of gene expression, growth, and differentiation. Although morphogenesis has been extensively studied from a molecular point of view, how genetic networks are translated into biological forms is still unclear. Thus, the aim of this research project is to study the development of lateral organs (rosette leaves, cauline leaves, and floral organs, i.e. sepals, petals, and anthers) in the model species Arabidopsis thaliana. To address the question of the role of complex cell-organ interactions during development, we are interested not only in variability between organs but also in the intrinsic cellular variability of each organ. We, therefore, tested (1) whether the diversity of shapes observed in lateral organs results from modulations of a common developmental program; (2) whether the growth and development of lateral organs is a stochastic phenomenon or depends on specific underlying mechanisms. To this end, we are using a multidisciplinary approach based on genetics, confocal microscopy, and 3D image analysis to extract the growth patterns inherent in the different organs. The results of the first study (Chapter 2) show that organ shape depends on the balance between growth and differentiation, which fine regulation enables the acquisition of highly specialized functions. The cauline leaf, for example, shows a delay in differentiation that allows for prolonged morphogenetic activity and growth redistribution. Through the transient growth suppression at early stages, the cauline leaf developmental trajectory allows for its dual function, from protection to photosynthesis. The second study (Chapter 3) focuses on the behavior of individual cells, whose growth, although controlled by positional information, is often heterogeneous. This variability results from the differentiation of specialized cells, the stomata, which follow a specific developmental program. The autonomous, asynchronous behavior of these cells is the main source of variability in tissues whose growth is otherwise homogeneous. Overall, this thesis has shed light on the importance of timing in plant organ development. Whether at the organ, tissue, or cell level, modulation and synchronization of growth and differentiation are necessary for the acquisition of stereotypic organ shapes and functional complexity.