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Ponts entre les séquences d'acides nucléiques et les protéines, les ribosomes sont des composants essentiels des cellules vivantes. Composé d'ARN et de protéines ribosomiques, ils sont transportés, durant leurs biogenèses, du nucléole au cytoplasme, où ils traduisent les ARN messagers (ARNm) en protéines. Ces dernières années,  il a été montré que nombre de protéines ribosomiques étaient impliquées dans le développement d'Arabidopsis en intervenant sur la division et l'élongation cellulaire. L'impact d'un défaut de biogenèse des ribosomes sur le développement pourrait être expliqué par un effet dose, par une spécificité des ribosomes pour leur ARNm cibles ou par la multifonctionnalité de protéines ribosomiques. Les résultats obtenus montrent que REBELOTE (RBL), l'un des deux homologues chez Arabidopsis de la protéine NOC2p de levure, intervient probablement durant la biogenèse des ribosomes. Des mutations dans le gène RBL causent une gamme de phénotype de la létalité embryonnaire aux défauts de croissance (réduction de la taille de la plante, altération de la forme des feuilles...). Afin de mieux comprendre les processus contrôlés par RBL, la fonction ribosomique de RBL a été étudiée et ses interacteurs protéiques recherchés. Nous nous sommes ensuite focalisé sur les effets des mutations rbl sur la division et l'élongation cellulaire. Ce travail montre que les défauts observés aux niveaux moléculaire et cellulaire peuvent expliquer les retards de croissance des mutants rbl. / Bridges between nucleic acids sequences and proteins, ribosomes are central components and the “auletes” of living cells.  Composed of ribosomal proteins and RNA, they move during their biogenesis from the nucleolus to the cytoplasm, where they translate RNA messengers into proteins. In the past years, some mutants of ribosomal-biogenesis-related proteins have shown the importance of these proteins during cell division and Arabidopsis development. The impact of ribosomal defects on development could be explained by dose effect (which could be important for cell fitness), specificity of ribosomes for some mRNA or multifunctional ribosomal proteins (Mary E. Byrne, 2009). Here I present our work on REBELOTE (RBL), one of the two Arabidopsis homologs of the yeast NOC2 protein, which act during the ribosomal 60S subunit biogenesis. Mutations in REBELOTE gene cause a range of phenotypes, from embryo lethality to growth defects (reduced plant size, altered leaf shape…). To have a better understanding of RBL-controlled processes, we first analyzed the ribosomal function of RBL, and searched for its protein partners. Our results shows that RBL act in two different nucleolar complexes supposed to regulate 60S ribosomal subunit biogenesis. Subsequently, we focused on the effects of rbl mutations on the cell division/elongation processes. Our work shows that defects observed at molecular and cellular levels could explain the slow down of cell divisions and growth delay in rbl mutants.

The natural genetic variation among A. thaliana populations in Catalonia was used to identify local adaptation to coastal and inland habitats. A Species Distribution Model (SMD) was created to locate multiple small stands of A. thaliana (demes). Results using 425 genome-wide SNP markers under clustering and population analysis indicate a high percentage of shared alleles among demes. Multi-year field-based reciprocal transplant experiments were designed to identify fitness trade-offs between inland and coastal demes. Progenies from these demes performed better in their local/home environments. Similar results were obtained in greenhouse common garden experiments, confirming that soil is a driving factor for local adaptation. Plants from the coastal habitat outperformed those from inland when grown together under high salinity. It is concluded that A. thaliana is locally adapted to coastal environments, and this adaptation is driven, at least in part, by the elevated salinity of coastal soils. Our results do not point to a single mechanism of salinity tolerance. AtSOS1 and AtHKT1;1 may cooperate increasing leaf Na+ and its vacuolar storage achieving better osmotic adjustment. Crossings between coastal (salt tolerant) and inland (salt sensitive) plants suggest maternal inheritance of salt tolerance. Polymorphisms in both AtHKT1;1 and AtMOT1 may be of adaptive significance because the weak alleles were only detected in coastal demes. However, all results indicate that genetic variability in AtHKT1;1 allele is not responsible for the salinity tolerance. We conclude that the weak allele of AtHKT1;1 persists and coexists with plants bearing the strong allele thanks to early flowering and better tolerance of moderate salinity. Moreover, the weak allele of AtMOT1 was more frequent and was detected nearer to the sea than the weak allele of AtHKT1;1. Results with mot1 knockout mutants under NaCl treatments indicate that loss of function of AtMOT1 may enhance tolerance to salt stress.

RNAs endógenos competitivos (ceRNAs) são transcritos naturais que atuam como esponjas endógenas de miRNAs, modulando a ação de miRNAs sobre mRNAs-alvo. RNA circular (circRNA) é uma dentre as várias classes de ceRNA. circRNAs são produzidos a partir de um processo chamado backsplicing. CircRNAs já foram identificados em diversos eucariotos; no entanto, nas plantas, ainda não foi demonstrado se estes são capazes de atuar como esponjas eficazes de miRNAs. Dados públicos de RNAseq de bibliotecas de imunoprecipitação de Argonauta (AGO-IP) a partir de flores de Arabidopsis thaliana foram utilizados em um método multi-comparativo de análises de bioinformática para identificar potenciais RNAs circulares. Utilizando cinco diferentes métodos, foram preditos de 15 a 27812 circRNAs com pelo menos dois reads na junção do backsplicing. Posteriormente, a plataforma psRNAtarget foi utilizada para discriminar os circRNAs com sítios de ligação de miRNAs, que potencialmente possam estar atuando como esponjas. Como a AGO forma um complexo ternário com miRNA e mRNA-alvo, também foram comparadas as contagens de alvos em bibliotecas AGO-IP e controle, demonstrando que os mRNAs-alvo desses miRNAs também estão enriquecidos nas bibliotecas AGO-IP. Neste trabalho, foram encontrados dois prováveis circRNAs que podem potencialmente funcionar como esponjas de miRNA. Esse achado contribui para um melhor entendimento desta nova forma de regulação transcricional e pós-transcricional em plantas, ampliando o conhecimento e aplicações do tema. / Competing endogenous RNAs (ceRNAs) are natural transcripts that act as endogenous sponges of miRNAs, modulating the action of miRNAs on mRNAs targets. Circular RNA (circRNA) is one among the various classes of ceRNA. They are produced from a process called backsplicing. CircRNAs have been identified in several eukaryotes; however, in plants, it has not yet been demonstrated whether they are able to act as effective sponges for miRNAs. Public RNAseq data from Argonaute immunoprecipitation libraries (AGO-IP) from Arabidopsis thaliana flowers were used with a multi-comparative method of bioinformatics analyzes to identify putative circular RNAs. Using five different methods, 15 to 27812 circRNAs with at least two reads at the backsplicing junction were predicted. Subsequently, the psRNAtarget platform was used to discriminate circRNAs harboring miRNA binding sites, which could potentially be acting as sponges. Identities and amounts of mRNAs present in AGO-IP and control libraries were quantified, as AGO can also form a ternary complex among miRNA and their target mRNAs. It showed that target mRNAs from circRNA:miRNA pairs were also enriched in the AGO-IP libraries. Two circRNAs were positively identified, corroborating their action as potential miRNA sponges. This finding leads to a better understanding of a new form of transcription and post-transcription regulation in plants, increasing the knowledge and applications of the topic.

Um dos grandes desafios da pesquisa agrícola atual é desenvolver estratégias para obtenção de plantas mais tolerantes, portanto, continuar ampliando o conhecimento sobre os mecanismos pelos quais as plantas respondem à seca, é essencial para a identificação de rotas metabólicas envolvidas no processo de defesa e o desenvolvimento de genótipos cada vez mais adaptados. Neste trabalho, para a obtenção de plantas de soja tolerantes à seca, a construção RD29A:AtDREB1A a qual confere tolerância ao déficit hídrico foi inserida em cultivares de soja e a biossegurança alimentar da proteína DREB1A/CBF3 de A. thaliana foi avaliada. Também, os extratos protéicos das plantas submetidas ao estresse hídrico foram comparados para a identificação de proteínas de soja diferencialmente expressas que podem estar envolvidas nas respostas à seca. A avaliação de biossegurança alimentar da proteína AtDREB1A in silico mostrou que a proteína não possui características de toxicidade, alergenicidade, antinutricionais, ou sítios de N-glicosilação, assim como, de atividade hemolítica sob eritrócitos de humanos atestado com a proteína AtDREB1A produzida in vitro, indicando ausência de efeitos adversos. A inserção da construção RD29A:AtDREB1A e RD29A:GUS em soja, demonstrou que o promotor RD29A e o fator de transcrição AtDREB1A de A. thaliana são ativados aumentando a tolerância ao déficit hídrico em soja. Vinte linhagens transgênicas foram obtidas por biobalística e apresentaram estabilidade do transgene. Análises histoquímicas confirmaram a indução do promotor RD29A em condições de desidratação e o aumento de expressão de genes de soja GmPip1 GmGols regulados pela proteína DREB1A também foi confirmado por RT-qPCR. Diferenças anatômicas significativas não foram observadas. Em média, os parâmetros fisiológicos foram superiores nas linhagens transgênicas, quando comparadas à sua isolinha BR16. Embora características agronômicas mais adaptativas relacionadas à produção não foram evidentes em casa de vegetação, há uma boa indicação de que a estratégia pode melhorar a tolerância à seca em plantas de soja. Para isso, experimentos a campo já estão sendo conduzidos para uma melhor caracterização agronômica e fisiológica. Na busca por ampliar os conhecimentos sobre os eventos envolvidos nas respostas à seca, a comparação dos extratos protéicos de soja BR16 não transgênica e BR16(P58) geneticamente modificada (GM) com a construção RD29A:AtDREB1A em condições controle e sob déficit hídrico, possibilitou a identificação de “spots” comuns aos dois tratamentos assim como de “spots” diferencialmente expressos, através da metodologia de 2-DE. Nos tratamentos de déficit hídrico, 25 “spots” foram identificados em BR16 não GM e 34 “spots” em P58 GM, entretanto, análises de espectrometria de massa ainda estão em andamento e pesquisas em bancos dados serão fundamentais para a identificação das proteínas diferencialmente expressas em resposta ao déficit hídrico. / One of the challenges in agriculture is to develop strategies to obtain plants with higher drought tolerance. Therefore, the identification of metabolic pathways and understanding of the mechanisms by which plants respond to drought is an essential tool for the development of more adapted genotypes. In this work, the RD29A:AtDREB1A genetic construct which was reported to confer water deficit tolerance was inserted into soybean cultivars, and the food safety of Arabidopsis thaliana AtDREB1A/CBF3 protein was evaluated. Also, protein extracts from soybean plants submitted to water stress conditions were compared to identify differentially expressed soybean proteins involved in drought responses. The in silico evaluation of AtDREB1A protein showed that the protein has no evidence of toxicity, allergenicity, antinutritional features or N-glycosylation sites. No hemolytic activity on human erythrocytes assayed in vitro with AtDREB1A protein was detected, indicating no adverse effects. The insertion of RD29A:AtDREB1A and RD29A:GUS constructs in soybean showed that the RD29A stress-inducible promoter and the AtDREB1A transcription factor were activated and improved drought tolerance in soybean. Using bioballistic transformation, we obtained twenty stably transformed soybean lines. Histochemical analysis confirmed the induction of the RD29A promoter under dehydration conditions and increased expression of two soybean genes activated by AtDREB1A GmPip1 and GmGols were confirmed by RTqPCR. No anatomical differences were observed. On average, physiological parameters were superior in the transgenic line BR16(P58) when compared to the isoline BR16. Although agronomic traits related to higher adaptive production were not evident in greenhouse, there is a good indication that the strategy can improve drought tolerance in soybean. To establish trait efficacy, field experiments are already being conducted to obtain relevant agronomic and physiological data. To better understand the molecular events involved in drought responses, a comparison using protein extracts from genetically modified (GM) BR16(P58) and non-GM soybean BR16 in watered control conditions and under water deficit treatment allowed the identification of common proteins to both treatments as well as of proteins that are differentially expressed between treatments using 2-dimension gel electrophoresis (2-DE). In the water deficit treatment 25 differentially expressed protein spots were identified in non-GM isoline BR16 and 34 spots in BR16(P58). Subsequent analysis of these spots using mass spectrometry is still been conducted and peptide information in available public databases will be essential for identification of differentially expressed proteins.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 123-132). Also available on the World Wide Web.

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 161-172). Available also in a digital version from Dissertation Abstracts.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy