Graduation date: 1993

Thesis (Ph. D.)--University of Oregon, 2002. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 174-182).

The mechanisms that genes use to direct patterns of development are of fundamental interest. Using Arabidopsis thaliana as a model, I have investigated aspects of these mechanisms in the separate processes of vascular and floral development. Specifically, I conducted a screen for vascular-defective mutants, and analyzed a region of the genome that regulates the expression of the floral homeotic gene, AGAMOUS ( AG). / In this report, I describe the identification of over forty mutants that are abnormal in tracheary element development or vein patterning. The spectrum of mutant phenotypes that I observed indicates that the mechanisms that pattern primary and secondary veins of leaves or cotyledons are at least partially separable; that among the genes that affect vascular development, a significant proportion are repressors of vascular differentiation; and that the majority of vascular mutants that can be identified in this type of screen have pleiotropic phenotypes. / I characterized two of the mutants, varicose ( vcs) and scarface (sfc), in more detail. vcs mutants are temperature sensitive, and at the non-permissive temperature, accumulate distended tracheary elements around veins. VCS is also required at an early stage of leaf development for normal vein patterning, and interacts with the AUXIN RESISTANT 1 gene in this process. sfc mutants fail to develop normal, contiguous vein networks in cotyledons, leaves, sepals, and petals. It is specifically the secondary and higher order veins in these organs that are affected by the mutation. sfc mutants have exaggerated responses to exogenous auxin, and the SFC gene overlaps in primary and secondary vein patterning functions with an auxin-response factor gene MONOPTEROUS. / This report also includes an analysis of the cis-regulatory regions that control expression of AGAMOUS, a gene that when properly expressed in two central domains of the developing flower, directs the formation of carpels and stamens. My dissection of an AG intragenic region demonstrated that AG expression in stamens can be activated independently of carpels. Moreover, the stamen-specific expression pattern was found to be independent of APETALA2, a known negative regulator of AG, while the carpel-specific expression pattern was shown to be independent of LEUNIG, another negative regulator of AG.

[Truncated abstract] Mitochondria are semi-autonomous organelles found in almost all eukaryotic cells to contain more than 1000 different proteins. The majority of these proteins are encoded in the nucleus, translated in the cytosol and imported into mitochondria. The overall aim of this study was to characterise the regulation of nuclear-encoded mitochondrial proteins (NEMP). This was carried out in the plant, Arabidopsis thaliana, using the alternative oxidase (AOX) as a model. Specifically, the aims were to i) determine how regulation of NEMP interact with known regulatory pathways/mechanisms; ii) determine if the pattern of coexpression observed for NEMP are due to co-regulation, and iii) to determine whether mitochondrial retrograde regulatory pathways interact with known chloroplast regulatory pathways. AOX1c is one of five genes encoding AOX in Arabidopsis. It is expressed in a variety of organs and is not induced by stress. Thus, its regulation was characterised in order to gain insight into the regulation of NEMP under normal growth conditions. Analysis of the promoter of AOX1c revealed cis-acting regulatory elements (CAREs) common to both AOX1c from Arabidopsis and AOX2b from soybean. Additionally, Site II elements, previously shown to be involved in the regulation of the proliferating cell nuclear antigen, are present in the upstream promoter region of AtAOX1c and were shown to be strong negative regulators of AtAOX1c expression. AOX1a is a gene encoding AOX that is induced at a transcript level, by many stress treatments. BA signalling and provide evidence of at least one common factor between chloroplastic and mitochondrial retrograde regulatory pathways, i.e. ABI4. ... The above results reveal that the regulation of NEMP are integrated with the mainstream regulatory pathways that control gene expression for a variety of proteins in various locations. Although this is not unexpected, it does raise the question of how mitochondrial function impacts, or feeds back, to alter these pathways, i.e. how mitochondrial retrograde signals affects the regulation of genes encoding proteins in a variety of locations. The observed interaction of mitochondrial and plastid retrograde regulatory pathways at the level of ABI4, suggests that mitochondrial signals have the potential to act as a powerful regulators of many cellular functions. Although interaction between mitochondrial and other organelles at a cellular level has been known for some time, there is still much work left to be done to define the network of molecular interactions that exists to regulate and integrate the expression of NEMP with all other proteins in the cell. This study reveals that interactions also occur at regulatory steps that have to potential to regulate many function in organelles, even if no direct metabolic link exists. However, this study has only begun to uncover these interactions at a molecular level.

L'agriculture mondiale a connu de profonds changements qui lui ont permis de faire face à l'augmentation constante de la demande alimentaire. Cependant, les conséquences de ces nouvelles pratiques agricoles sur l'environnement et la santé humaine font l'objet de préoccupations croissantes. Notamment, les politiques sanitaires actuelles visent à réduire l'utilisation des produits phytosanitaires. Aussi de nouvelles stratégies de protection des cultures doivent-elles être développées. Une meilleure compréhension des échanges moléculaires qui contribuent au succès des bioagresseurs est nécessaire. Ces échanges impliquent notamment la sécrétion de protéines qui interfèrent avec le métabolisme de l'hôte : les effecteurs. Certains d'entre elles sont accumulés au cours de la pénétration des premières cellules végétales, une étape décisive pour le succès de la tentative d'infection. Les travaux menés au cours de cette thèse se sont concentrés sur 3 de ces effecteurs, sécrétés par l'oomycète Phytophthora parasitica. L'analyse des lignées de surexpression chez Arabidopsis thaliana a permis de mettre en évidence des perturbations du développement et de la physiologie de certaines hormones végétales en réponse à l'accumulation de ces effecteurs. Ces données confirment l'importance de la manipulation des voies hormonales dans le cadre des interactions plantes-pathogènes et soutiennent l'hypothèse récente selon laquelle des effecteurs sécrétés par les agents pathogènes interfèrent avec un petit nombre de cibles clefs du métabolisme de l'hôte. Ces cibles constituent des candidats de choix pour développer des variétés plus résistantes. / Agriculture has undergone deep changes that have allowed to cope with the ever-increasing demand for food. However, the consequences of the new agricultural practices on the environment and human health are the subject of increasing concern. Notably, current health policies aimed at reducing the use of pesticides in agriculture. Thus, new strategies need to be developed for efficient crop protection. In particular, a better understanding of molecular exchanges that contribute to the success of pathogens is required. These exchanges include the secretion of proteins that interfere with host metabolism : the effectors. Some of them are accumulated during the penetration of the first plant cells, a crucial step for the success of the infection attempt. This thesis work focused on three of these effectors, secreted by the oomycete Phytophthora parasitica. The analysis of transgenic Arabidopsis thaliana lines highlighted perturbations of plant development and hormone physiology in response to the accumulation of these effectors. These data confirm the pivotal role of hormonal balance during plant-pathogen interactions and support the recent hypothesis that effectors secreted by evolutionarily distant plant pathogens interfere with a small number of key target host metabolism. These targets are good candidates to develop varieties that are more resistant to infection.

Les télomères sont des structures nucléoprotéiques spécialisées qui assurent l’intégrité des extrémités des chromosomes linéaires. Ils sont composés d’une séquence d’ADN qui leur est propre, sont associées à des protéines spécifiques, et sont maintenus par la télomérase. Leur fonction est d’empêcher le raccourcissement progressif des extrémités des chromosomes dû à la réplication ainsi que leur reconnaissance par les mécanismes de réparation des cassures double brin. La réparation des cassures double-brin consiste en deux mécanismes généraux : la recombinaison non-homologue et la recombinaison homologue. L’activation de ces voies de recombinaison en réponse à la déprotection télomérique est un mécanisme de survie cellulaire, mais qui conduit fréquemment à une instabilité génomique.Nous avons testé l’implication des voies de recombinaison non-homologue dans la prise en charge des télomères déprotégés par inactivation de chacune de ces voies. La génération d’un multiple mutant ne présentant pas de voies de recombinaison non-homologue fonctionnelles a permis de restaurer la capacité des plantes à se développer normalement en présence de télomères déprotégés. Nos résultats ont mis en évidence une compétition entre les voies de recombinaison non-homologue et la télomérase en absence de protection télomérique.L’étude des homologues de la protéine GEN1 – impliquée dans la résolution de structures d’ADN branchées lors de la recombinaison homologue – a permis d'identifier l'homologue fonctionnel de GEN1 chez Arabidopsis et de mettre en évidence un rôle télomérique de cette protéine. En effet, cette nucléase s’avère essentielle à la stabilité des télomères en favorisant leur réplication.L’inhibition de la recombinaison homologue en absence de télomérase entraine l’apparition précoce et significative d’instabilité chromosomique. L’induction d’un stress réplicatif exacerbe cette instabilité télomérique, indiquant que la recombinaison homologue facilite la réplication des télomères en absence de télomérase. Nos données indiquent que la télomérase participe également au déroulement correct de la réplication des télomères. De façon inattendue, le rôle positif de la recombinaison homologue est dépendant de l’hélicase RTEL1. / Telomeres are specialized nucleoprotein structures that ensure the integrity of the ends of linear eukaryotic chromosomes. They are composed of a particular DNA sequence, associated with specific proteins and are maintained by telomerase. Their function is to prevent the progressive shortening of chromosome due to replication and to protect chromosome ends from recognition by cellular double-strand break repair proteins. Double-strand break repair involves two general mechanisms: non-homologous recombination and homologous recombination. Activation of these recombination pathways in response to unprotected telomeres is a cell survival mechanism, which frequently leads to genomic instability.We tested the involvement of non-homologous recombination pathways in fusions of unprotected telomeres by inactivation of each of these pathways. The generation of a triple KO mutant without non-homologous recombination restores the normal growth and development of the plants with deprotected telomeres. These results show a competition between non-homologous recombination pathways and telomerase in the absence of telomeric protection.Study of GEN1 homologues – involved in the resolution of branched DNA structures during homologous recombination – permitted identification of the functional GEN1 homologue of Arabidopsis and the demonstration of a telomeric role of this protein. Indeed, this nuclease proves to be essential to the stability of the telomeres by promoting their replication.Inhibition of homologous recombination in absence of telomerase leads to the early and significant appearance of chromosomal instability. Induction of replicative stress exacerbates this telomere instability, indicating that homologous recombination facilitates telomere replication in the absence of telomerase. Our data indicate that telomerase also contributes to correct replication of telomeres. Unexpectedly, the positive role of the homologous recombination in telomere stability is dependent on the RTEL1 helicase.

L'auteur n'a pas fourni de résumé en français / L'auteur n'a pas fourni de résumé en anglais

L’ARN hélicase UPF1 est un facteur clé du Nonsense-Mediated Decay (NMD), un mécanisme impliqué dans le contrôle de la qualité des ARNm et la régulation de l’expression des gènes. Malgré d’importantes fonctions chez les plantes, le NMD y est peu décrit. Cette thèse présente l’identification et l’étude des protéines interagissant avec UPF1 chez Arabidopsis. Nous avons identifié un nouveau réseau d’interaction protéine-protéine entre UPF1 et des répresseurs de traduction dans les P-bodies. Nous proposons un modèle dans lequel la répression traductionnelle exerce une action protectrice sur les cibles du NMD. Notre approche a également identifié de nouveaux composants des P-bodies, comme l’endonucléase UCN. Son étude détaillée a révélé un lien direct avec la machinerie de decapping ainsi que de possibles rôles dans la signalisation hormonale ou les mécanismes de défense, suggérant que la modulation de l’expression d’UCN pourrait influencer d’importantes caractéristiques agronomiques. Ce travail décrit des facteurs associés à UPF1 jusqu’alors inconnus, leur étude permettra de découvrir de nouveaux mécanismes impliqués dans l’équilibre entre la traduction, le stockage et la dégradation des ARNm chez les plantes. / The RNA helicase UPF1 is a key factor of Nonsense-Mediated Decay (NMD), a paneukaryotic mechanism involved in mRNA quality control and fine-tuning of gene expression. Despite important biological functions in plants, NMD is poorly described compared to other eukaryotes. This thesis presents the identification and study of UPF1 interacting proteins in Arabidopsis. Using approaches based on immunoaffinity and mass spectrometry, we identified a novel protein-protein interaction network between UPF1 and translation repressors in P-bodies. We propose a model in which translation repression exerts a protective action on NMD targets in plants. Our approach also identified novel P-body components, including the UCN endonuclease. A detailed study revealed its direct link with the decapping machinery and possible roles in hormone signaling and defense mechanisms, suggesting that the modulation of UCN expression could influence important agronomical traits. This work describes hitherto unknown UPF1 associated factors, their study will provide novel insights into the mechanisms involved in the balance between mRNA translation, storage and decay in plants.

La ppGpp et la pppGpp, qui sont synthétisées/hydrolysées par les RelA/Spot homologs (RSH), jouent un rôle centrale dans l’adaptation des bactéries contre la privation des nutriments et les stress environnementaux. Les enzymes RSH et ppGpp ont été découverts dans le chloroplaste. Il a été récemment démontré que ppGpp joue un rôle comme répresseur globale de l’expression de gènes chloroplastiques. Certains stresses environnementaux et hormones induisent l’accumulation de ppGpp chez les plantes, cependant le mécanisme moléculaire n’est pas encore connu. Ici nous nous sommes intéressés à découvrir les facteurs qui interagissent avec les RSH, et qui donc sont susceptible de réguler le métabolisme du ppGpp. En utilisant un crible double-hybridation de levure nous avons identifiées des proteines qui interagissent avec les RSH y compris l’acyl carrier protein (ACP) et des GTPases associées au ribosome. ACP et RSH1 semblent être indispensables pour l’accumulation de ppGpp induite par la carence de la biosynthèse des acides gras, tandis que le ppGpp et un GTPase associé au ribosome contribuent à la résistance contre le heat-shock. Nous avons aussi effectué du co-immunoprécipitation spectrométrie de masse avec RSH1. Plusieurs protéines ont été identifiées y compris des protéines associées au nucléoid et des protéines liées à la signalisation chloroplastique, indiquant que RSH1 pourrait etre impliqué dans la machinerie de transcription chloroplastique. Nos résultats montrent que chez les plantes le ppGpp joue un rôle non seulement comme chez les bacteriés mais aussi participe à de nombreux processus biologiques qui sont spécifiques aux plantes. / Guanosine tetra-phosphate and penta-phosphate (ppGpp and pppGpp), which are synthesized/hydrolyzed by RelA/Spot homolog (RSH) enzymes, play a central role in the adaptation of bacteria to nutrient limitation or other stresses. Both RSH enzymes and ppGpp are present in the chloroplasts of plants. Recent studies have shown that ppGpp acts as a global repressor of chloroplast gene expression. Certain environmental stresses and hormones induce ppGpp accumulation in chloroplasts, however the molecular mechanisms underlying the activation of ppGpp signalling in response to such stimuli is essentially unknown. We searched for factors that interact with RSH enzymes and so could play a role in activating ppGpp signalling. Using a targeted yeast two hybrid screen several proteins were identified that interact with RSH enzymes including acyl carrier protein (ACP) and ribosome associated GTPases. ACP and RSH1 appear to be required for ppGpp induction in response to fatty acid biosynthesis depletion, while ppGpp and an RSH-interacting GTPase contribute to the resistance of plants to heat shock. We also performed non-targeted co-immunoprecipitation mass spectrometry (CoIP-MS) of RSH1. New RSH interaction candidates were identified, including plastid nucleoid associated proteins and chloroplast signalling proteins, suggesting that RSH1 may be associated with the plastid transcription machinery. Our results give new insights into ppGpp signalling, and show that some elements are conserved between plants and bacteria, while others are implicated in plant-specific biological processes.

Submitted by MARCOS LEANDRO TEIXEIRA DE OLIVEIRA (marcosteixeira@ufv.br) on 2018-09-06T11:28:25Z No. of bitstreams: 1 texto completo.pdf: 2396350 bytes, checksum: d4a4b3c74df2af6f3609cee963d570e2 (MD5) / Made available in DSpace on 2018-09-06T11:28:25Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2396350 bytes, checksum: d4a4b3c74df2af6f3609cee963d570e2 (MD5) Previous issue date: 2018-07-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A via de sinalização de morte celular mediada por proteínas NRP/DCDs foi primeiramente descrita em soja como uma via integrativa de resposta a múltiplos estresses. A falta de ferramentas moleculares adequadas à caracterização dessa via em soja levou ao estudo de sua conservação em espécies modelo. Em Arabidopsis, os membros da via foram descritos, no entanto não foi definido o ortólogo do gene GmNAC30. Neste trabalho, foi conduzida uma análise in silico da subfamília SNAC-A (ATAF), à qual pertence GmNAC30, demonstrando ser ela amplamente distribuída no reino vegetal. Em comparação com Arabidopsis, foi verificada uma expansão dessa subfamília em soja, o que pode estar relacionado à pressão seletiva que espécies cultiváveis sofrem a favor da manutenção de genes capazes de conferir tolerância a estresses. Consistente com essa ideia, a análise da expressão de alguns membros desse subgrupo em soja demonstrou que todos eles respondem a múltiplos estresses. Os genes duplicados da subfamília SNAC-A em soja exibiram padrão e cinética de indução por polietilenoglicol (PEG) e Tunicamicina (TUN) similares; porém, os pares apresentaram diferenças sutis de indução por AS, indicando que devem possuir funções parcialmente sobrepostas, mas não absolutamente idênticas. A fim de verificar a hipótese difundida de ser ATAF1 o ortólogo de GmNAC30 foram realizados ensaios de interação de ATAF1 com ANAC036 (ortólogo de GmNAC081) pelo sistema de duplo-híbrido de leveduras e análises de expressão gênica de ATAF1 em resposta a estresses osmótico e no retículo endoplasmático (RE). Os resultados obtidos, no entanto, indicaram não ser ATAF1 o ortólogo de GmNAC30, uma vez que não interagiu com ANAC036 pelo sistema de duplo- híbrido, e ao contrário de GmNAC030, não foi induzido por tunicamicina (TUN), embora, assim como GmNAC030, ATAF1 tenha exibido atividade transcricional pelo sistema de mono-híbrido de leveduras. Uma possível interação do gene ATAF1 com a via de morte celular induzida por estresses no RE e osmótico foi avaliada por genética reversa no mutante ataf1-2 que exibiu 75% de redução na expressão de ATAF1. Tanto a expressão basal quanto a expressão induzida por PEG e TUN dos genes NRP-1 e ANAC036, componentes da via de morte celular, foi superior no mutante ataf1-2 do que em Col-0, implicando ATAF1 como possível regulador negativo da via de morte celular mediada por NRPs. Sendo ATAF2 um outro membro do subgrupo SNAC-A que está filogeneticamente muito próximo a GmNAC30, foi avaliada a sua resposta a tratamentos com PEG e tunicamicina em plântulas das linhagens Col-0 e ataf1-2. Foi observada a indução de ATAF2 em resposta a ambos os estresses, sendo que a indução é aumentada no mutante ataf1-2, consistente com o perfil de expressão gênica de integrantes da via de morte celular mediada por NRPs. Coletivamente, estes resultados substanciam o argumento de que ATAF2 possa ser ortólogo de GmNAC030. No entanto, experimentos complementares são necessários para a confirmação dessa hipótese. / The NRP/DCDs- mediated cell death signaling was first described in soybean as an integrative pathway for multiple stresses. The lack of adequate molecular tools for characterization of this pathway in soybean led to the demonstration of its conservation in plant model systems. In Arabidopsis, several members of this family have been described, but the GmNAC30 ortholog remains to be identified. In this investigation, an in silico analysis of the SNAC-A (ATAF) subfamily of GmNAC30 was conducted and demonstrated that SNAC-A is largely distributed in the plant kingdom. As compared to Arabidopsis, the SNAC-A subfamily was expanded in soybean, more likely due to the selective pressure undergone by economically relevant crops towards the maintenance and duplication of stress related genes. Accordingly, an expression analysis of some soybean genes of this subfamily demonstrated that they respond to multiple stresses. The duplicated genes from the soybean SNAC-A subfamily displayed similar profile and induction kinetics by PEG and tunicamycin (TUN). Nevertheless, the pairs exhibited slightly differences in salicylic acid (SA) induction, indicating that they may display partially overlapping, but not absolutely identical functions. To confirm that ATAF1 is a GmNAC030 ortholog, yeast two-hybrid assays for ATAF1-ANAC036 (GmNAC081 otrholog) interaction and analysis of ATAF1 gene expression in response to osmotic and endoplasmic reticulum (ER) stresses were conducted. However, the results indicated that ATAF1 is not likely a GmNAC30 ortholog, as ATAF1 did not interact with ANAC036 in yeast and, opposing to the GmNAC030 expression profile, it was not induced by TUN, although like GmNAC030, ATAF1 was transcriptionally active by the yeast mono-hybrid assay. A possible interaction of ATAF1 with the ER stress- and osmotic stress-induced cell death signaling was monitored by reverse genetic in ataf1-2 mutant, which displayed a 75% reduction in the accumulation of ATAF1 transcripts. Both basal and stress-induced expressions of NRP-1 and ANAC036, components of the cell death pathway, were higher in ataf1-2 than in Col-0, implicating ATAF1 as a possible negative regulator of the NRPs- mediated cell death signaling. Because ATAF2 is another member of the SNAC-A subfamily, which is phylogenetically close related to GmNAC30, its response to PEG and TUN was monitored in Col-0 and ataf1-2 seedlings. ATAF2 was induced by both treatments and the transcript accumulation was further increased in ataf1-2, which was consistent with the expression profile of components of the NRPs-mediated cell death signaling. Collectively, these results substantiate the argument that ATAF2 may be a GmNAC030 ortholog. However, complementary experiments are necessary to confirm this hypothesis.