Les interactions entre l’auxine et l’éthylène sont complexes et contrôlent divers processus de développement des plantes tels que l’élongation racinaire ou la différentiation des racines secondaires. Mais, il existe peu d’études montrant le rôle des interactions entre ces deux hormones au cours du développement et de la maturation des fruits. Le changement de couleur des fruits chez la tomate est une caractéristique de la maturation qui est associée à la fois à la dégradation des chlorophylles et à l’accumulation des caroténoïdes. Dans ce travail, l’application exogène d’auxine et d’éthylène a montré l’impact de ces deux hormones sur la maturation de la tomate et en particulier sur le changement de couleur des fruits. Nous avons montré que l’acide indol-acétique (IAA) retarde la transition du vert à l’orange/rouge, alors que l’éthylène, apporté sous la forme d’acide 1-aminocyclopropane-1-carboxylique (ACC), son précurseur, accélère la coloration des fruits. Par contre, l’inhibition de l’auxine par le PCIB, un antagoniste de l’auxine, provoque les mêmes effets que l’éthylène. L’analyse des caroténoïdes montre que l’ACC comme le PCIB augmente la teneur en lycopène et diminue la teneur en carotène alors que l’IAA provoque l’effet inverse. L’étude de l’accumulation des ARNs messagers de plusieurs gènes clés de la voie de biosynthèse des caroténoïdes a montré que le gène β-lcy codant pour la lycopène cyclase joue un rôle clé dans le contrôle de la biosynthèse et de l’accumulation des pigments et que son expression est fortement dépendante de l’équilibre auxine-éthylène. D’autre part, nos résultats ont montré que le gène rin joue un rôle important dans le contrôle de l’expression des gènes clés de la voie de biosynthèse des caroténoïdes. Pour avoir une meilleure vision des gènes différentiellement exprimés par l’auxine et l’éthylène au cours de la maturation, l’analyse du transcriptome des fruits traités par de l’ACC et de l’IAA a été réalisée par RNA-Seq au laboratoire. Parmi les facteurs de transcriptions étudiés, le gène Sl-IAA17, un membre de la famille des AUX/IAA, est fortement affecté par l’auxine et l’éthylène. La caractérisation fonctionnelle du gène Sl-IAA17 pendant le développement du fruit a été réalisée en créant des lignées transgéniques sous exprimant ce gène en mettant en œuvre la stratégie des ARNs interférents. Ces lignées présentent un phénotype caractéristique produisant des fruits de plus gros calibre que celui des fruits sauvages. Les analyses histologiques des tissus des fruits ont montré que ce phénotype est associé à un péricarpe plus épais. En microscopie, nous avons constaté que l’augmentation de l’épaisseur du péricarpe dans les lignées transgéniques n’était pas due à un plus grand nombre de cellules mais à l’augmentation de la taille des cellules. Enfin, nous avons observé que l’expansion des cellules dans les fruits transgéniques est étroitement couplée avec des niveaux de ploïdie plus élevés que dans les fruits sauvages, ce qui suggère une stimulation du processus endoréduplication. Ces résultats démontrent très clairement l’existence d’une étroite relation entre la signalisation de l’auxine, le contrôle de la taille du volume cellulaire et le processus d’endoréduplication. En conclusion, les résultats présentés fournissent des connaissances nouvelles sur les interactions entre l’auxine et l’éthylène au cours du développement du fruit et en particulier au cours de la transition fruit immature - fruit mature. De plus, ils apportent des éléments nouveaux sur la connaissance du rôle de la voie de signalisation de l’auxine dans le contrôle du développement des fruits charnus et en particulier sur la fonction de certains membres des AUX/IAA sur la détermination du volume et du poids des fruits. / The interaction between auxin and ethylene are complex and control various processes of plant development, such as root elongation or differentiation of secondary roots. But there are few studies showing the role of interactions between these two hormones during development and maturation of the fruit. The color change in the tomato fruit is a feature of the maturation that is associated with the degradation of the chlorophyll and carotenoid accumulation. In this work, the application of exogenous auxin and ethylene showed the impact of these two hormones in the tomato ripening and in particular the change of fruit color. We have shown that indole-acetic acid (IAA) delays the transition from green to orange / red, while ethylene, supplied as 1-aminocyclopropane-1-carboxylic acid form (ACC), its precursor, accelerated this transition. However the auxin inhibition by p-chlorophenoxy isobutyic acid (PCIB), an auxin antagonist, caused the same effects similar to ethylene. The carotenoid analysis showed that the ACC and PCIB increase the lycopene content and reduced the carotene content while IAA causes the opposite effect. The study of the accumulation of mRNAs for several key genes of the carotenoid biosynthetic pathway has shown that the gene β-lcy encoding lycopene cyclase plays a key role in the control of biosynthesis and accumulation of pigments and that its expression is highly dependent on the auxin-ethylene balance. In addition, our results showed that the rin gene plays an important role in controlling the expression of the key carotenoid biosynthetic pathway genes. To get a better view of differentially expressed genes by auxin and ethylene during ripening, transcriptome analysis of fruits treated with ACC and IAA was performed by a preliminary RNA-Seq approach. Among the transcription factors studied in the laboratory, the gene Sl-IAA17, a member of the family of Aux/IAA was affected by auxin and ethylene. Functional characterization of Sl-IAA17 gene during fruit development was performed by creating transgenic lines under-expressing this gene by RNAi. These lines display a phenotype producing bigger fruit than wild type. Histological analysis of the tissues showed that fruit phenotype is associated with a thicker pericarp. By microscopy, we observed that increasing the thickness of the pericarp in the transgenic lines was not due to a greater number of cells but to the increase in cell size. Finally, we observed that cell expansion in transgenic fruit is tightly coupled with higher ploidy levels than wild fruits, suggesting a stimulation of the endoreduplication process. These results clearly demonstrate the existence of a close relationship between the auxin signal, the control cell size, fruit volume and the endoreduplication process. In conclusion, the results provide new insights into the interactions between auxin and ethylene during fruit development and in particular during the transition immature fruit, mature fruit. In addition, they provide new information on the understanding of the role of the signaling pathway of auxin in controlling the development of fleshy fruits and in particular on the basis of certain members of the AUX/IAA on regulating volume and fruit weight.

A eficiência da reprodução sexual de plantas depende do correto desenvolvimento dos órgãos sexuais: estame e pistilo. Mecanismos moleculares complexos controlam a proliferação e expansão celular que resultam no correto desenvolvimento destes órgãos. Em nosso laboratório foi identificado um gene preferencialmente expresso no pistilo de Nicotiana tabacum, o gene 092H06. Este gene codifica uma pequena proteína de 68 aminoácidos, e função desconhecida. Análises anteriores sugerem que o produto proteico do gene 092H06 seja responsável por inibir o processo de expansão celular nos órgãos reprodutivos (Brito,2010). Para compreender o papel deste gene, no desenvolvimento do pistilo, foram realizados experimentos de qRT-PCR para determinar se os níveis de expressão de genes para -expansina, -expansina, ciclina B1.2 e actina, ligados aos processos de divisão e expansão celular, em plantas transgênicas de silenciamento e superexpressão do gene 092H06. Foram realizadas análises morfológicas nos estigmas/estiletes e ovários das plantas transgênicas de segunda geração (T2), por microscopia óptica. Os resultados mostram uma tendência de aumento no volume das células tanto nas plantas transgênicas de silenciamento, como nas de superexpressão. Entretanto, nas plantas de silenciamento ocorreu um aumento visível das estruturas reprodutivas, o que não foi observado nas plantas de superexpressão. Adicionalmente, foram realizados experimentos de citometria de fluxo, para verificar a ocorrência de endorreduplicação. Os resultados mostraram que não ocorreu endorreduplicação nas células das plantas transgênicas. No screening de uma biblioteca de duplo híbrido, usando 092H06 como isca, foram encontrados 4 candidatos a parceiros de interação: 1) biotin/lipolyl attachmente domain-containing protein; 2) unknown protein; 3) trypsin proteinase inhibitor precursor e 4) RING/U-box. Para auxiliar no estudo da função do gene 092H06, a proteína recombinante 092H06-Histag foi produzida com sucesso, na forma solúvel, em E. coli. Os resultados alcançados neste trabalho contribuem para avançar o conhecimento sobre este novo gene expresso nos órgãos reprodutivos das plantas. / The efficiency of plant sexual reproduction depends on the correct development of the sexual organs: stamen and pistil. Complex molecular mechanisms control cell proliferation and expansion that result in the correct development of these organs. In our laboratory a gene preferentially expressed in Nicotiana tabacum pistil has identified, the 092H06 gene. This gene encodes a small protein of 68 amino acids of unknown function. Previous analyzes suggest that the protein product of the gene 092H06 is responsible for inhibiting the cell expansion process in the reproductive organs (Brito, 2010). To understand the role of this gene in pistil development, experiments of qRT-PCR to determinate the expression levels of the -expansins, -expansins, cyclin B1.2 and actin, genes which connected to the cell division and expansion processes, were carried out on transgenic plants silencing and overexpressing the 092H06 gene. Morphological analyzes on stigmas/styles and ovaries of second generation (T2) transgenic plants were performed by optical microscopy. The results show a tendency to increased cellular volume on the silencing transgenic plants, as well as on the overexpressing plants. However, in the silencing plants there was a visible increase of the reproductive structures, what has not been observed on the overexpressing plants. Additionally, flow cytometry experiments were carried out to verify the occurrence of endoreduplication. The results showed that no endoreduplication has occurred on the cells of the transgenic plants. The screening of a yeast two-hybrid assays, using 092H06 as bait, has found 4 interaction partners candidates: 1) biotin/lipolyl attachment domain-containing protein; 2) unknown protein; 3) trypsin proteinase inhibitor precursor and 4) RING/U-box. To assist the study of the 092H06 function, the recombinant 092H06-HIStag protein has been produced with success, in the soluble form, in E.col. The results obtained in this work contribute to advance the knowledge of this novel gene expressed on the plant reproductive organs.

A eficiência da reprodução sexual de plantas depende do correto desenvolvimento dos órgãos sexuais: estame e pistilo. Mecanismos moleculares complexos controlam a proliferação e expansão celular que resultam no correto desenvolvimento destes órgãos. Em nosso laboratório foi identificado um gene preferencialmente expresso no pistilo de Nicotiana tabacum, o gene 092H06. Este gene codifica uma pequena proteína de 68 aminoácidos, e função desconhecida. Análises anteriores sugerem que o produto proteico do gene 092H06 seja responsável por inibir o processo de expansão celular nos órgãos reprodutivos (Brito,2010). Para compreender o papel deste gene, no desenvolvimento do pistilo, foram realizados experimentos de qRT-PCR para determinar se os níveis de expressão de genes para -expansina, -expansina, ciclina B1.2 e actina, ligados aos processos de divisão e expansão celular, em plantas transgênicas de silenciamento e superexpressão do gene 092H06. Foram realizadas análises morfológicas nos estigmas/estiletes e ovários das plantas transgênicas de segunda geração (T2), por microscopia óptica. Os resultados mostram uma tendência de aumento no volume das células tanto nas plantas transgênicas de silenciamento, como nas de superexpressão. Entretanto, nas plantas de silenciamento ocorreu um aumento visível das estruturas reprodutivas, o que não foi observado nas plantas de superexpressão. Adicionalmente, foram realizados experimentos de citometria de fluxo, para verificar a ocorrência de endorreduplicação. Os resultados mostraram que não ocorreu endorreduplicação nas células das plantas transgênicas. No screening de uma biblioteca de duplo híbrido, usando 092H06 como isca, foram encontrados 4 candidatos a parceiros de interação: 1) biotin/lipolyl attachmente domain-containing protein; 2) unknown protein; 3) trypsin proteinase inhibitor precursor e 4) RING/U-box. Para auxiliar no estudo da função do gene 092H06, a proteína recombinante 092H06-Histag foi produzida com sucesso, na forma solúvel, em E. coli. Os resultados alcançados neste trabalho contribuem para avançar o conhecimento sobre este novo gene expresso nos órgãos reprodutivos das plantas. / The efficiency of plant sexual reproduction depends on the correct development of the sexual organs: stamen and pistil. Complex molecular mechanisms control cell proliferation and expansion that result in the correct development of these organs. In our laboratory a gene preferentially expressed in Nicotiana tabacum pistil has identified, the 092H06 gene. This gene encodes a small protein of 68 amino acids of unknown function. Previous analyzes suggest that the protein product of the gene 092H06 is responsible for inhibiting the cell expansion process in the reproductive organs (Brito, 2010). To understand the role of this gene in pistil development, experiments of qRT-PCR to determinate the expression levels of the -expansins, -expansins, cyclin B1.2 and actin, genes which connected to the cell division and expansion processes, were carried out on transgenic plants silencing and overexpressing the 092H06 gene. Morphological analyzes on stigmas/styles and ovaries of second generation (T2) transgenic plants were performed by optical microscopy. The results show a tendency to increased cellular volume on the silencing transgenic plants, as well as on the overexpressing plants. However, in the silencing plants there was a visible increase of the reproductive structures, what has not been observed on the overexpressing plants. Additionally, flow cytometry experiments were carried out to verify the occurrence of endoreduplication. The results showed that no endoreduplication has occurred on the cells of the transgenic plants. The screening of a yeast two-hybrid assays, using 092H06 as bait, has found 4 interaction partners candidates: 1) biotin/lipolyl attachment domain-containing protein; 2) unknown protein; 3) trypsin proteinase inhibitor precursor and 4) RING/U-box. To assist the study of the 092H06 function, the recombinant 092H06-HIStag protein has been produced with success, in the soluble form, in E.col. The results obtained in this work contribute to advance the knowledge of this novel gene expressed on the plant reproductive organs.

Les légumineuses sont une source riche pour l’alimentation humaine comme celle du bétail mais elles sont aussi nécessaires à une agriculture durable. Cependant, les fractions majeures des protéines de réserve dans la graine sont pauvres en acides aminés soufrés et peuvent être accompagné de facteurs antinutritionnels, ce qui affecte leur valeur nutritive. Dans ce cadre, Medicago truncatula est une espèce modèle pour l’étude du développement de la graine des légumineuses, et en particulier concernant la phase d’accumulation des protéines de réserve. Vu la complexité des graines de légumineuses, une connaissance approfondie de leur morphogenèse ainsi que la caractérisation des mécanismes sous-jacents au développement de l’embryon et au remplissage de la graine sont essentielles. Une étude de mutagenèse a permis d’identifier le facteur de transcription DOF1147 (DNA-binding with One Finger) appartenant à la famille Zn-finger, qui s’exprime dans l’albumen pendant la transition entre les phases d’embryogenèse et de remplissage de la graine. Lors de mon travail de thèse, il a été possible de générer plusieurs constructions pour l’analyse de l’expression de DOF1147 ainsi que de la protéine DOF1147. Un protocole efficace pour la transformation génétique stable de M. truncatula a été établi et des études de localisation subcellulaire ont montré que DOF1147 est une protéine nucléaire. Un arbre phylogénétique a révélé différents groupes de facteurs de transcription DOF avec des domaines conservés dans leur séquence protéique. L’analyse du promoteur in silico chez plusieurs gènes-cible potentiels de DOF1147 a identifié les éléments cis-régulateurs de divers facteurs de transcription ainsi que des éléments répondant aux auxines (AuxREs), ce qui suggère un rôle possible de l’auxine pendant le développement de la graine. Une étude in vitro du développement de la graine avec divers régimes hormonaux, a montré l’effet positif de l’auxine sur la cinétique du développement de la graine, que ce soit en terme de gain de masse ou de taille, plus fort avec l’ANA que l’AIB. Grâce à une approche cytomique de ces graines en développement nous avons, en plus, démontré l’effet de l’auxine sur la mise en place de l’endoreduplication. En effet, celle-ci est l’empreinte cytogénétique de la transition entre les phases de division cellulaire et d’accumulation de substances de réserve lors du développement de la graine. Dans son ensemble, ce travail a démontré que l’auxine module la transition entre le cycle mitotique et les endocycles chez les graines en développement de M. truncatula en favorisant la continuité des divisions cellulaires tout en prolongeant simultanément l’endoreduplication. / Legumes are not only indispensible for sustainable agriculture but are also a rich source of protein in food and feed for humans and animals, respectively. However, major proteins stored in legume seeds are poor in sulfur-containing amino acids, and may be accompanied by anti-nutritional factors causing low protein digestibility problems. In this regard, Medicago truncatula serves as a model legume to study legume seed development especially the phase of seed storage protein accumulation. As developing legume seeds are complex structures, a thorough knowledge of the morphogenesis of the seed and the characterization of regulatory mechanisms underlying the embryo development and seed filling of legumes is essential. Mutant studies have identified a DOF1147 (DNA-binding with One Finger) transcription factor belonging to the Zn-Finger family which was expressed in the endosperm at the transition period between embryogenesis and seed filling phase. During my PhD work, a number of transgene constructs were successfully generated for expression analysis of DOF1147 gene as well as the DOF1147 protein. A successful transformation protocol was also established for stable genetic transformation of M. truncatula. Subcellular localization studies have demonstrated that DOF1147 is a nuclear protein. A phylogenetic tree revealed different groups of DOF transcription factors with conserved domains in their protein sequence. In silico promoter analysis of putative target genes of DOF1147 identified cis-regulatory elements of various transcription factors along with auxin responsive elements (AuxREs) suggesting a possible role of auxin during seed development. A study of in vitro seed development under different hormone regimes has demonstrated the positive effect of auxin on kinetics of seed development in terms of gain in seed fresh weight and size, with NAA having a stronger effect than IBA. Using the cytomic approach, we further demonstrated the effect of auxin on the onset of endoreduplication in such seeds, which is the cytogenetic imprint of the transition between the cell division phase and the accumulation of storage products phase during seed development. As a whole, this work highlighted that the auxin treatments modulate the transition between mitotic cycles and endocycles in M. truncatula developing seeds by favouring sustained cell divisions while simultaneously prolonging endoreduplication.