Diversity in plant architecture is largely generated by the post-embryonic regulation of meristem initiation and activity. In a phenomenon known as apical dominance, the active growth of the shoot apical meristem (SAM) exerts significant inhibitory force on the outgrowth of axillary meristems (AMs) into shoot branches. The degree of branching in plants is a determinant of yield in many crop species and is carefully regulated to ensure that plants only branch at specific stages of development or in response to their environment. Apical dominance has been attributed to the action of the hormone auxin, produced in SAM tissues and transported downwards. A second hormone, cytokinin, acts antagonistically to auxin to promote branching. Nonetheless, the exact mechanism by which these hormones operate is still being elucidated and continued research suggested that novel signals are involved. The recent discovery that strigolactones, previously implicated in parasitic weed germination and mycorrhizal associations, are branching inhibitors supports the existence of additional signals controlling branching in plants. In garden pea (Pisum sativum) strigolactones are synthesised by the coordinated action of the carotenoid cleavage dioxygenase (CCD) family enzymes, RMS1 (RAMOSUS1) and RMS5. These are encoded by MAX4 (MORE AXILLARY GROWTH4) and MAX3 in Arabidopsis thaliana respectively. Mutants for MAX genes have increased amounts of auxin travelling in the polar auxin transport stream (PATS) of inflorescence stems but exhibit increased branching that is insensitive to inhibition by this auxin. Two hypotheses for the action of strigolactones have been presented. The first is that strigolactones modulate the levels of auxin transport proteins, preventing axillary buds from establishing an active auxin transport flow into the primary stem, which inhibits growth. The second is that strigolactones act downstream of auxin signalling to inhibit the action of outgrowth-promoters. Consistent with this latter hypothesis, in pea, rice (Oryza sativa) and petunia (Petunia hybrida), the expression of RMS1/MAX4 orthologues is auxin regulated. These genes are also regulated by feedback signalling in strigolactone pathway mutants and this is proposed to involve an additional novel signal. In Arabidopsis, however, research showed that MAX4 is not regulated by feedback or auxin in the shoot and placed doubt on the importance of this regulation for branching control. The strigolactone biosynthetic pathway offers a novel target for the manipulation of plant architecture and yield while controlling the germination of parasitic weed species that are detrimental to agriculture. Therefore, a greater understanding of the pathway and its regulators is beneficial. The majority of the research in this thesis pre-dates the discovery of strigolactones as the RMS/MAX-derived branching inhibitor, yet aimed to clarify the evolutionary conservation and functional importance of the regulation of strigolactone biosynthetic genes by auxin and feedback signalling in Arabidopsis. Quantitative real-time PCR analysis demonstrated that MAX3 and MAX4 are co-ordinately and systemically regulated by auxin and by feedback throughout development. Both auxin and feedback regulation required the AXR1/TIR1 auxin response pathway, which targets Aux/IAA transcriptional repressors for proteasomal degradation. In particular, correct degradation of the Aux/IAA protein IAA12 appears to be necessary for optimal MAX3 and MAX4 expression. Moreover this regulation affects strigolactone-dependent branching inhibition. Therefore it is proposed that auxin inhibits branching, in part, by positively regulating strigolactone synthesis. As feedback requires AXR1, this also suggests that increased auxin level and/or signalling in the PATS in conditions of reduced strigolactone signalling mediates feedback regulation of the strigolactone pathway. Consistent with this, microarray analysis revealed that in addition to the inflorescence, max mutants have increased global auxin-responsive gene expression associated with the PATS in the vegetative stage. The pea RMS1 gene was the first strigolactone pathway gene demonstrated to be auxin-regulated. Sequencing of the RMS1 promoter and comparative bioinformatic analysis with promoters of other strigolactone synthesis genes revealed a number of conserved, putative regulatory cis-elements that could mediate this regulation and cross-talk with additional branching cues. However a 2.5 kb fragment of the RMS1 promoter was not sufficient to drive transcriptional and translational fusions with GFP and the RMS1 coding region in Arabidopsis. The RMS1 coding region driven by the CAMV 35S promoter complemented the max4 mutant but did not affect branching induced by auxin-depleting treatments. Grafting studies with axr1 and iaa12 mutants, and decapitation and auxin-transport inhibition in max4 mutants, demonstrated that auxin signalling has a function in branching control independent from the regulation of strigolactone synthesis genes. Overall, data obtained herein was incorporated into current models for the interaction of the strigolactone pathway with auxin and cytokinin in the control of shoot branching. It is suggested that both strigolactone and auxin have the capacity to regulate the levels or distribution of each other in interlocking feedback loop that intersects with additional developmental, physiological and environmental cues for the precise control of axillary branching in plants.

Os hormônios auxina e citocininas são essenciais ao desenvolvimento das plantas, pois controlam os processos de divisão, expansão e diferenciação celular, os quais, por sua vez, influenciam desde a formação do embrião até o amadurecimento dos frutos e senescência. Auxinas e citocininas regulam respostas fisiológicas comuns, sugerindo haver múltiplos mecanismos de interação. Neste trabalho, um modelo para estudar a interação entre auxinas e citocininas no controle do desenvolvimento é proposto, sendo baseado em plantas mutantes e transgênicas, além de duplos mutantes, com alterações na sensibilidade ou metabolismo de auxina e citocinina. O mutante bushy root (brt) foi caracterizado como pouco sensível à citocinina e sugere uma importante função da citocinina no desenvolvimento da semente e na determinação da dominância apical em tomateiro. Os mutantes potato leaf (c) e entire (e) foram introgredidos no background Micro-Tom (MT), caracterizados e utilizados para verificar como eles afetam a sensibilidade à auxina e como interferem no desenvolvimento da planta. Esses mutantes foram comparados com MT, com o mutante diageotropica (dgt), que é pouco sensível à auxina, e com os duplos mutantes c dgt e dgt e. A mutação c não alterou a sensibilidade à auxina e estaria influenciando apenas a arquitetura foliar. O mutante e parece ser mais sensível à auxina com relação à capacidade de formar raízes in vitro e partenocarpia. O duplo mutante dgt e mostrou fenótipo aditivo (intermediário), sugerindo que DGT e E agem em vias paralelas na resposta à auxina. Foram criadas linhagens transgênicas com superexpressão da enzima citocinina oxidase de Arabidopsis (35S:AtCKX2), que resulta em plantas com baixos níveis endógenos de citocinina. As linhagens transgênicas (MT CKX2 e dgt CKX2) foram comparadas com MT, brt, dgt e brt dgt. A maioria dos parâmetros que estavam relacionados com o desenvolvimento vegetativo, e todos os parâmetros relacionados ao desenvolvimento reprodutivo, foram associados ao efeito dos níveis absolutos de auxina e citocinina, sendo provavelmente resultantes de alterações nos processos de divisão e expansão celular. A dominância apical e a morfogênese in vitro (formação de gemas caulinares e raízes) foram associadas ao efeito do balanço auxina/citocinina, sendo esses processos conhecidamente regulados pelo efeito dessas classes hormonais na diferenciação celular / The plant hormones auxin and cytokinin are crucial for plant development, since they control cell division, expansion and differentiation, which regulate developmental processes starting from embryo formation to fruit ripening and senescence. Auxins and cytokinins usually regulate the same physiological responses, suggesting multiple mechanisms of interaction between these hormones. In this work, a model to study auxin and cytokinin interaction in the control of plant development is proposed, based on mutants, transgenic lines and double mutants with alterations on the auxin and cytokinin sensitivity or metabolism. The bushy root (brt) mutant was characterized as having low cytokinin sensitivity and suggested a relevant function to cytokinin in the seed development and apical dominance in tomato. The potato leaf (c) and entire (e) mutants were introgressed in the Micro-Tom background, characterized and used to check how they affect the auxin sensitivity and the plant development. The responses from these mutants were compared to MT and diageotropica (dgt), which is a low sensitive auxin mutant, and also compared to c dgt and dgt e double mutants. The c mutation did not alter the auxin sensitivity and was only related to leaf architecture. The e mutant seemed to be more sensitive to auxin in the in vitro root induction and parthenocarpy. The double mutant dgt e showed an additive phenotype, suggesting that DGT and E act in parallel pathways controlling auxin sensitivity. Transgenic lines were generated overexpressing the cytokinin oxidase from Arabidopsis (35S:AtCKX2), which renders plants with low endogenous cytokinin levels. Transgenic lines (MT CKX2 and dgt CKX2) were compared to MT, brt, dgt and brt dgt. Most of the traits related to vegetative development and all traits related to reproductive development were linked to the effect of auxin and cytokinin absolute levels, probably reflecting to the effect of these hormones in cell division and expansion. Apical dominance and in vitro morphogenesis (shoot and root formation) were linked to the auxin-to-cytokinin ratio, since these processes are well known to be regulated by the effect of these two hormones in cell differentiation

Submitted by EDER MARQUES DA SILVA null (biodida@yahoo.com.br) on 2016-05-23T19:54:50Z No. of bitstreams: 1 TESE_Eder30 04.pdf: 2779899 bytes, checksum: c45da4458d84767b126f9e3b3af87767 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-05-25T18:03:50Z (GMT) No. of bitstreams: 1 silva_em_dr_bot.pdf: 2779899 bytes, checksum: c45da4458d84767b126f9e3b3af87767 (MD5) / Made available in DSpace on 2016-05-25T18:03:50Z (GMT). No. of bitstreams: 1 silva_em_dr_bot.pdf: 2779899 bytes, checksum: c45da4458d84767b126f9e3b3af87767 (MD5) Previous issue date: 2016-05-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O processo de frutificação é definido como a transição de um ovário em estágio quiescente para estágio de iniciação de formação do fruto. Esta transição é iniciada em resposta aos eventos de polinização e fertilização, os quais induzem o início dos processos fisiológicos e moleculares, que, ao final, irão originar o fruto. A frutificação é fundamental para reprodução nas plantas com flores (Angiospermas). Já foi demonstrado que hormônios (tais como auxina e giberelina) podem atuar em paralelo com fatores de transcrição durante o desenvolvimento do fruto. Alguns desses fatores de transcrição são pós-transcricionalmente regulados por microRNAs. MicroRNAs, são pequenos RNAs (20-22 nt) que regulam pós-transcricionalmente a expressão de genes endógenos, modelando o transcriptoma e a produção de proteínas.Entretanto, vias genéticas orquestradas por microRNAs associadas com o desenvolvimento de ovário, e consequentemente, do fruto em tomateiro (Solanum lycopersicum L.), permanecem pouco exploradas. Neste trabalho, foi investigada a contribuição da via SlymiR159/GAMYB-like durante o processo de desenvolvimento do ovário e frutificação em tomateiro. O miR159 e seus alvos (SlyGAMYB1 e 2) são dinamicamente expressos durante os estágios de desenvolvimento do ovário e consequentemente do fruto. Eventos transgênicos de tomateiro (cv. Micro-Tom) apresentando super-expressão do precursor SlyMIR159 (denominados OE-159) exibiram frutificação precoce quando comparadas aos controles. Notavelmente, todos os eventos OE-159 apresentaram formação de frutos partenocárpicos. Tal alteração no padrão de frutificação pode estar correlacionada a repressão do gene SlyGAMYB1 em períodos pré-antese de desenvolvimento do ovário, é possível que a atividade do fator de transcrição SlyGAMYB1 seja importante para prevenir o início da frutificação antes da polinização. Além disso, a via regulada pelo miR167/SlyARF8A mostrou-se desregulada em ovários pré-antese de plantas transgênicas OE-159. Adicionalmente, mesmo com o fenótipo de partenocarpia e frutificação precoce, não houve alterações na quantidade de hormônios (e.g. giberelina, ácido abscisico e auxina) relacionados à formação de frutos nas plantas transgênicas OE-159. Contudo, a via miR159/GAMYB-like encontra-se reprimida nos primeiros estágios da frutificação (após polinização e fertilização) e a expressão do SlyARF8A (alvo do miR167) encontra-se induzida na presença de auxina. Em conjunto, nossos dados sugerem que o crosstalk entre as vias moduladas pelos microRNAs miR159 e miR167, integrados com a via de sinalização de auxina, é importante para a frutificação em tomateiro. / Fruit set, defined as the shift from quiescent ovary to a fast-growing young fruit, is a key process for fruit production in flowering plants. It has been shown that hormones (such as auxin and gibberellin) act in parallel with transcription factors during fruit set. Some of these are post-transcriptionally regulated by microRNAs (miRNAs). MiRNAs are a group of small non coding RNAs (21-22 nt) that act by regulating post-transcriptionally genes in plants and animals However, microRNA-regulated genetic pathways associated with tomato (Solanum lycopersicum L.) ovary development and fruit set remain poorly exploited. Here, we investigated the functional role of miRNA159/SlyGAMYBlike in tomato ovary development and fruit set. MiR159 and its targets were dynamically expressed in developing flowers, ovaries and fruit tissues. Transgenic tomato (cv. Micro-Tom) plants over-expressing the SlyMIR159 (termed OE-159) exhibited fruit set earlier when compared with control plants. Strikingly, all transgenic lines presented parthenocarpy fruits. Such developmental modification may be a result of the repression of SlyGAMYB1 in pre-anthesis ovaries, our data suggest that SlyGAMYB1 activity is important to prevent fruit set before pollination. In addition as a consequence of this repression, the miR167 node (miR167/SlyARF8 pathway) was mis-regulated in OE-159 ovaries. In addition, we observed that levels of auxin, gibberellin (GA) and abscisic acid (ABA) in pre-anthesis ovaries of several transgenic lines overexpressing the SlyMIR159 were similar to those of the control. Moreover, our experiments suggested that auxin and GA may act repressing the miR159 node during fruit set initiation, in order to release the miR167-targeted SlyARF8A expression during fruit set. Our data show how two microRNA nodes (miR159 and miR167) integrated with hormone signalling into a circuit that coordinates successive steps along ovary and fruit development.

Resumo: Plântulas provenientes da germinação de sementes in vitro da bromélia Aechmea blanchetiana foram cultivadas em meio líquido para o estudo do crescimento sob a influência de diferentes reguladores vegetais. Os tratamentos consistiram da combinação de concentrações (mg L-1) de BAP (0,2), Kt (0,2), NAA (0,1) e IBA (0,1) adicionados aos meios de cultivo líquido MS. As plântulas foram cultivadas sob fotoperíodo de 16 horas e temperatura de 25 ºC ± 2, durante 90 dias e subcultivos a cada 15 dias. O delineamento experimental adotado foi o inteiramente casualizado para todas as variáveis consideradas com 9 tratamentos e 4 repetições, totalizando 36 parcelas, sendo cada parcela composta por 4 plântulas perfazendo um total de 144 plântulas. Foram medidas e analisadas as seguintes características: comprimento da maior folha, número de folhas, número de raízes, comprimento da maior raiz, massa fresca e seca de plântulas e raízes. O BAP promoveu o melhor resultado para as variáveis número de raízes e comprimento da maior raiz. Os tratamentos com NAA e IBA apresentaram o melhor resultado quanto ao comprimento da maior folha, gerando ambos melhores desempenho para a massa de matéria fresca de plântulas / Abstract: Seedlings from in vitro seed germination of the bromeliad Aechmea blanchetiana were cultured in liquid medium to study growth under influence of different plant growth regulators. Treatments were combined concentrations (mg L-1) of BAP (0.2), Kinetin (0.2), NAA (0.1) and IBA (0.1) added to MS liquid culture media. Seedlings were cultured under 16h photoperiod and 25 ºC ± 2 for 90 days and subcultured every 15 days. Experimental design was completely randomized for all variables, with 9 treatments and 4 replicates totaling 36 plots, each one composed of 4 seedlings, 144 in total. Length of the largest leaf, number of leaves, number of roots, length of the largest root, and fresh and dry matter of seedlings and roots were measured and analyzed. BAP led to the best results for number of roots and length of the largest root. Treatments with NAA and IBA led to the best values as to length of the largest leaf, both resulting in higher values of seedling fresh matter / Orientador: Elizabeth Orika Ono / Coorientador: Márcio Christian Serpa Domingues / Banca: João Domingos Rodrigues / Banca: Armando Reis Tavares / Mestre

Resumo: O cancro cítrico é uma doença de importância mundial para o cultivo de laranjas e seu agente causador é a bactéria Xanthomonas axonopodis pv. citri. Embora todos os genes presentes nessa bactéria já tenham sido sequenciados, grande parte destas sequências não apresenta qualquer informação experimental sobre a contribuição para o sucesso da instalação do patógeno na planta hospedeira. Por outro lado, torna-se cada vez mais evidente que várias bactérias fitopatogênicas utilizam mecanismos de adaptação de origem genética que podem induzir efeitos fisiológicos na planta e adequar o ambiente de tal forma que permita a ocorrência da doença. Dentre estes mecanismos, está a possibilidade da bactéria produzir substâncias que se assemelhem a hormônios vegetais, como a auxina (AIA). O objetivo deste trabalho foi desenvolver metodologia genômica de busca para investigar se há genes que codificam para a produção de compostos iguais ou semelhantes a este hormônio em X. axonopodis pv. citri e se a sua participação no processo de patogênese é fundamental para o sucesso da infecção. Para tal, foi produzida coleção de mutantes genéticos funcionais aleatoriamente em X. axonopodis pv. citri, com posterior submissão desta mesma coleção a teste de produção de auxina utilizando o reagente Salkowski. Após produção e análise de grupo de mutantes dirigidos e coleção de aproximadamente 14200 mutantes aleatórios não foi detectada linhagem defectiva para a produção de AIA ou composto semelhante. Os resultados obtidos neste estudo, no entanto, mostram que a biossíntese do composto em X. axonopodis pv. citri é dependente do triptofano e que existe uma concentração ótima deste aminoácido a ser adicionado no meio de cultura para que a bactéria realize a biossíntese. Este é o primeiro estudo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The citrus canker is a disease of global significance for the cultivation of oranges and its causative agent is the bacterium Xanthomonas axonopodis pv. citri. Although all the genes present in this bacterium have been sequenced, most of these sequences have no experimental information regarding the contribution to the successful installation of the pathogen on the host plant. On the other hand, it has been clearly demonstrated that several pathogenic bacteria use adaptative mechanisms that can induce physiological effects in plants and adjust the environment in a way that allows the occurrence of the disease. Among these mechanisms there is the possibility the bacteria produce substances that are similar to plant hormones such as auxin (IAA). The objective of this study was to develop a screening methodology to investigate the presence of genes that code for a compound identical or similar to auxins in X. axonopodis pv. citri as well as if it plays a role during the pathogenesis process. The main goal of this study was to produce a collection of random and directed genetic mutant strains of X. axonopodis pv. citri for the screening of auxin production by using the Salkowski reagent. After production and analysis of the group of mutants, including a collection of approximately 14,200 random mutant strains, defective production of IAA or similar compound was not detected. On the other hand, the results of this study show that such compound mimics effects of hormonal properties in X. axonopodis pv. citri and is dependent of tryptophan. In addition, it is shown that there is an optimum concentration of this amino acid to be added in the culture medium for the bacteria to carry out biosynthesis. To our knowledge, this is the first study to investigate systematically the production of auxin-like compounds in Xanthomonas axonopodis pv. citri. / Orientador: Alexandre Morais do Amaral / Coorientador: Henrique Ferreira / Banca: Nelson Wulff / Banca: Dario Abel Palmieri / Mestre

I investigated two aspects of in vivo splicing that are poorly understood: spliceosome disassembly and recycling, and proofreading. To this end, I used the auxin-inducible degron (AID) to individually deplete several splicing factors in budding yeast and then I measured the effect on co-transcriptional spliceosome assembly through chromatin immunoprecipitation. In addition, using RNA next-generation sequencing, I measured the frequency of splicing errors following depletion or mutation of the fidelity factor, Prp22. I show that formation of the pre-spliceosome (the first stage of spliceosome assembly) is rapidly inhibited by global defects in late stages of spliceosome assembly. I demonstrate that this is due to the accumulation of arrested spliceosomes that sequester the splicing machinery and, as a result, causes a recycling defect. This suggests that spliceosomes that lack essential splicing factors are not always properly disassembled and recycled in vivo, and warns about potential systematic secondary effects when perturbing single components of the spliceosome. Secondly, I describe the development of a new version of the AID system for budding yeast, called the B-estradiol AID. To the best of my knowledge, an AID system for budding yeast that is fast-acting, tightly-controlled and gratuitous, was lacking until now. Lastly, I show that absence of Prp22 protein, which was previously proposed to play a role in splicing fidelity, correlates with more mistakes in 3’ss selection of many endogenous intron-containing transcripts in vivo. This provides indirect evidence to suggest that Prp22-dependent splicing proofreading is physiologically important. The data from this analysis will be useful in ongoing studies to try to identify common features that could improve our understanding of the mechanism of Prp22’s function in splicing proofreading.

The diploma thesis was aimed on role of polar auxin transport in regulation of cotyledonary bud outgrowth in pea plants (Pisum sativum L.) in relation to competitive canalization of auxin sources. Decapitation of growth apex, as primary source of auxin, causes release of cotyledonary buds from inhibition. This was followed by increased expression of genes for auxin transport proteins PsPIN1 and PsAUX1 and decrease in PsDRM1 and PsBR1 gene expression that are related to dormant state of plant tissues. In the next phase, different types of exogenous auxin application on decapitated stumps and cotyledonary bud growth dynamic were carried out. Auxin application on decapiteded stump caused inhibition of cotyledonary bud growth, its application on right or left side of the stump inhibits cotyledonary bud on the side of application. If auxin on the both sides of stump was applied, cotyledonary bud growth was not inhibited, on contrary to their separation with a plastic sheet, which resulted in significant growth inhibition of both buds. In case of both-sided application, these sources of auxin prevents each other in canalization. If these two sources of auxin were separated with a plastic sheet, canalization of auxin flow was observed and two independent auxin channels were formed. Analogy of this competition can be expected in the case of cotyledonary buds, as two potential auxin sources.

O eucalipto é uma das espécies arbóreas mais plantadas no mundo atualmente, principalmente devido ao seu uso como matéria prima para as indústrias de celulose, papel e madeireira. Eucalyptus globulus e seus híbridos possuem baixos teores de lignina e despertam grande interesse da indústria, já que essa característica facilita e diminui o custo do processo de extração da celulose. Entretanto, essa espécie é recalcitrante ao enraizamento adventício, o que dificulta a propagação vegetativa de suas mudas. Com o objetivo de melhor entender os mecanismos moleculares envolvidos na recalcitrância ao enraizamento em E. globulus, o presente estudo envolveu análises de parâmetros morfológicos, anatômicos e moleculares durante a rizogênese adventícia nesta espécie. A exposição à auxina exógena reverteu o fenótipo recalcitrante em E. globulus, aumentando significativamente a porcentagem de enraizamento. O câmbio vascular foi identificado como uma região de acúmulo de auxina e local de origem das raízes adventícias. Através de análises da expressão gênica em células do câmbio, observou-se que TOPLESS e IAA12, repressores da sinalização de auxina, e ARR1, envolvido na rota de sinalização de citocininas, parecem atuar como reguladores negativos do enraizamento adventício. A alta expressão destes genes em plantas controle foi significativamente diminuída com aplicação de auxina exógena. Comparativamente, em espécie de fácil enraizamento, E. grandis, a expressão destes genes se manteve em níveis mais baixos em ambas as condições de tratamento, e a concentração de ácido indol-3-acético endógeno em plantas controle mostrou-se mais elevada. Análises do padrão proteico durante o enraizamento em plantas de E. globulus tratadas ou não com auxina exógena identificaram proteínas envolvidas em diversos processos biológicos, principalmente estresse oxidativo e metabolismo energético. Diferenças interessantes foram identificadas ao comparar as diferentes condições e fases do enraizamento. Várias proteínas foram claramente relacionadas com o respectivo fenótipo apresentado pela planta em cada situação, principalmente considerando plantas controle. Os resultados aqui apresentados representam avanços relevantes no conhecimento sobre a rizogênese adventícia em plantas lenhosas, podendo ser utilizados como ferramentas no desenho de estratégias visando melhorar o enraizamento em genótipos recalcitrantes de valor para a indústria. / Eucalyptus is one of the most planted tree species in the world today, mainly due to its use as raw material for paper, cellulose and wood industries. Eucalyptus globulus and its hybrids have low lignin contents and are of great interest to industry, as this feature facilitates and reduces costs of the cellulose extraction process. However, this species is recalcitrant to adventitious rooting, making vegetative propagation by cuttings difficult. Aiming at a better understanding of the molecular mechanisms involved in rooting recalcitrance in E. globulus, this study analyzed changes in morphological, anatomical and molecular patterns during adventitious rooting in this species. Exogenous auxin exposure reversed the recalcitrant phenotype in E. globulus, significantly increasing rooting percentage. The vascular cambium was identified as a region of auxin accumulation and also the site from where adventitious roots originated. Gene expression analysis in cambium cells indicated that TOPLESS and IAA12, auxin signaling repressors, and ARR1, involved in cytokinin signaling pathway, appear to act as negative regulators of adventitious rooting. The high expression of those genes in control plants was significantly decreased by exogenous auxin treatment. Comparatively, in an easy-to-root species, E. grandis, the expression of these genes was significantlylower in both treatment conditions, and the concentration of endogenous indole- 3-acetic acid in control plants was higher. Analysis of the protein pattern during rooting in E. globulus plants treated or not with exogenous auxin allowed the identification of proteins involved in diverse biological processes, mainly oxidative stress and energy metabolism. Interesting differences were identified when comparing different rooting conditions or phases. Several proteins were clearly associated with the respective plant phenotype in each situation, particularly considering control plants. These results represent relevant advances in the knowledge about adventitious rooting in woody plants and can be used as tools in the design of strategies aiming at improving adventitious rooting in recalcitrant genotypes of industrial value.

L'auxine est une hormone végétale impliquée dans le développement du gynécée. La réponse à l'auxine dépend majoritairement de deux familles protéiques : les ARF (Auxin Response Factors) et les Aux/IAA. Les ARF sont des facteurs de transcription qui régulent la transcription des gènes de réponse à l'auxine, tandis que les Aux/IAA inhibent l'activité transcriptionnelle des ARF par dimérisation. En présence d'auxine, les Aux/IAA sont dégradés et libèrent les ARF. Au cours de ma thèse, je me suis focalisée sur deux proches paralogues ETTIN/ARF3 et ARF4 impliqués dans le développement du gynécée chez Arabidopsis thaliana. Parmi les cibles directes d’ETT identifiées dans l'équipe, 2 catégories ont retenu notre attention : des gènes impliqués dans la voie de signalisation de l'auxine et des gènes codant des enzymes de remodelage de la paroi. Mon projet a consisté à essayer de comprendre l'implication de ces régulations transcriptionnelles dans le développement du gynécée. Dans une première partie, j'ai étudié la signalisation de l'auxine dans le développement du gynécée, puis l'implication d'ETT dans cette signalisation. Dans une deuxième partie, j'ai confirmé la répression par ETT, des cibles identifiées. Nous avons montré qu'ETT induit la déméthylation des pectines en inhibant les inhibiteurs de pectine méthylestérase. De plus, j'ai montré qu'ETT et ARF4 agiraient de façon redondante pour contrôler positivement la croissance du gynécée. Dans une troisième partie, j'ai abordé l'analyse des domaines fonctionnels du facteur de transcription CRABS CLAW, également impliqué dans le développement du gynécée. En conclusion, mes travaux de thèse apportent une nouvelle vision sur le rôle de la signalisation de l'auxine au cours du développement du gynécée et montrent l'importance du remodelage de la paroi dans la morphogénèse des organes. / The phytohormone auxin, is implicated in gynoecium development. Auxin response mainly depends on two protein families: ARFs (Auxin Response Factors) and Aux/IAAs. ARFs are transcription factors that regulate the transcription of auxin responsive genes, whereas Aux/IAAs inhibit the ARFs transcriptional activity by dimerization. In the presence of auxin, Aux/IAAs are degraded and consequently, ARFs are released. During my thesis, I focused on two close paralogs ETTIN/ARF3 and ARF4 involved in gynoecium development in Arabidopsis thaliana. Among the direct targets of ETT identified in the team, two categories held attention: genes involved in auxin signaling and genes encoding enzymes involved in cell wall remodeling. The aim of my project was to understand the implication of these transcriptional regulations in gynoecium development. In a first part, I analyzed auxin signaling during gynoecium development, as well as the implication of ETT in this pathway. In a second part, I confirmed the repression of the target genes by ETT. Interestingly, we showed that ETT activates the pectins demethylation by inhibiting the inhibitors of pectin methylesterase. Furthermore, I showed that ETT and ARF4 act in a redundant manner to positively control gynoecium growth. In a third part, I initiated the analysis of the functional domains of CRABS CLAW, a transcription factor also involved in gynoecium development. In conclusion, these results bring new information on the role of auxin signaling during gynoecium development and highlight the importance of cell wall remodeling in morphogenesis.

Philosophiae Doctor - PhD (Biotechnology) / The 'Bon Rouge' pear cultivar was developed from a bud mutation on a 'Bon Chretien' pear tree. The latter is characterised by green fruit skin and leaves, while 'Bon Rouge' is characterised by red leaves and red fruit skin as a result of the production of anthocyanin and other pigments. Branch forming buds on 'Bon Rouge' trees often revert to the parent phenotype producing green leaves and fruit skin. The occurrence of both phenotypes on the same tree presents a unique model to study gene expression associated with anthocyanin production in a similar genetic background under the same set of environmental condition.