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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.

Die durch den obligat biotrophen Protisten Plasmodiophora brassicae hervorgerufene Pflanzenkrankheit Kohlhernie verursacht weltweit hohe ökonomische Verluste. Bis heute gibt es keine effektiven Möglichkeiten, diese Pflanzenkrankheit zu bekämpfen. Eine Analyse der Genexpression in infizierten Wurzeln im Vergleich zu nicht infizierten Wurzeln ergab, dass die Gene für Lipid-Transfer-Proteine während der gesamten Krankheitsentwicklung differentiell reguliert sind. Über die Funktionen von Lipid-Transfer-Proteinen in Pflanzen wird noch spekuliert. Diskutiert wird dabei eine Funktion bei der Anpassung an verschiedene abiotische Stressfaktoren, bei der Pathogenabwehr sowie bei dem Transfer von Lipiden. In dieser Arbeit wurden transgene Pflanzen generiert, in denen die pathogenbedingte LTP-Genregulation umgekehrt ist. Es wurden transgene A. thaliana Pflanzen erzeugt, die die Gene LTP1, LTP3, LTP4, AT1G12090 sowie AT2G18370 überexprimieren und die Genexpression von AT4G33550 sowie AT1G62510 reprimieren. Die Regulation der LTP-Genexpression erfolgte dabei durch den wurzel- und keimlingsspezifischen Promotor Pyk10. Zusätzlich wurden in dieser Arbeit auch T-DNA-Insertionsmutanten für die Gene AT1G12090, AT2G18370, AT3G22620, AT5G05960, LTP3 sowie LTP4 untersucht. Mittels semiquantitativer Expressionsanalyse konnte die Modulation der LTP-Genexpression in den LTP-Mutanten bestätigt werden. Darüber hinaus konnte gezeigt werden, dass die Modulation der Expression eines LTP-Gens auch die Expression anderer LTP-Gene beeinflusst. Die phytopathologischen Analysen der LTP-Mutanten hinsichtlich der Entwicklung der Pflanzenkrankheit Kohlhernie ergab, dass die Überexpression der Gene LTP1, LTP3 sowie AT2G18370 und die Repression der Expression von AT1G62510 eine verringerte Anfälligkeit für diese Krankheit bewirkt. Die verstärkte Expression der Gene LTP1, LTP3, LTP4, AT1G12090 sowie AT2G18370 resultiert außerdem in einer verringerten Symptomentwicklung infolge einer Pseudomonas syringae-Infektion. Die verringerte Expression des Gens AT4G33550 führt hingegen zu einer größeren Anfälligkeit für eine P. brassicae Infektion; die Infektion mit P. syringae wird dadurch aber nicht beeinflusst. Die physiologische Charakterisierung der LTP-Mutanten umfasste die Analyse des Pflanzenwachstums unter Salzstress bzw. osmotischem Stress sowie die Entwicklung der Seneszenz in abgetrennten Rosettenblättern. Es konnte gezeigt werden, dass die Gene LTP1, LTP3, LTP4, AT4G33550 sowie AT1G62510 bei der Anpassung an Salzstress sowie die Gene LTP3, AT3G22620, AT4G33550 und AT1G62510 bei der Anpassung an osmotischen Stress eine Rolle spielen. Durch die Modulation der Expression der genannten Gene wird das Wachstum unter diesen Stressbedingungen sowohl positiv als auch negativ beeinflusst. Die Entwicklung der Seneszenz wird ebenfalls durch eine veränderte LTP-Genexpression (LTP1, LTP3, LTP4, AT3G22620 sowie AT4G33550) beeinflusst. Für die biochemische Charakterisierung wurden die LTP-Gene aus A. thaliana mit einem Fusionspartner in E. coli exprimiert und die resultierenden Fusionsproteine gereinigt. Diese wurden nach Abspalten des Fusionspartners hinsichtlich ihrer antimikrobiellen Aktivität und auf die Fähigkeit, Calmodulin zu binden, untersucht. Für die gereinigten Lipid-Transfer-Proteine LTP1, LTP3, LTP4, AT2G18370 sowie AT1G62510 konnte unter den bisher getesteten Versuchsbedingungen keine antimikrobielle Aktivität nachgewiesen werden. Für die Proteine LTP1, LTP3 und LTP4 konnte eine calciumunabhängige Calmodulin-Bindung nachgewiesen werden. Die Ergebnisse dieser Versuche ermöglichen keine Aussage bezüglich der genauen Funktion der einzelnen Lipid-Transfer-Proteine, geben aber Hinweise darauf, dass diese bei den entsprechenden Stress-Vorgängen eine Rolle spielen. Welche Funktion sie dabei genau erfüllen, muss in weiterführenden Analysen untersucht werden.

[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.

[Truncated abstract] A range of biotic and abiotic stresses increase levels of reactive oxygen species (ROS) in plants due to perturbations of chloroplast and mitochondrial metabolism and the generation of ROS in defence responses. The polyunsaturated fatty acids of membrane lipids are susceptible to ROS induced peroxidation yielding various aldehydes, alkenals and hydroxyalkenals including the cytotoxic compound 4-hydroxy- 2-nonenal (HNE). HNE has the potential to cause substantial oxidative damage in cells via its reactivity with sulfhydryl groups of cysteine (Cys) and lipoic acid, the imidazole group of histidine (His) and the ?-amino group of lysine (Lys) protein residues. Analysis of the components of the plant respiratory electron transport chain to HNE revealed a particular susceptibility to inhibition of activity of the alternative oxidase (Aox). Incubation with HNE prevented dimerisation of Aox protein, suggesting that one site of modification was the conserved cysteine residue involved in dimerisation and activation of this enzyme (Cys1). However, a naturally occurring isoform of Aox lacking Cys1 and unable to dimerise, LeAox1b from tomato, was equally sensitive to HNE inhibition, showing that other amino acid residues in Aox also interact with HNE and are likely responsible for inactivation of the enzyme. ... The broader impact of HNE on the whole Arabidopsis mitochondrial proteome was examined by use of various 2-dimensional gel separation techniques coupled with use of HNE-adduct antibodies. 32 proteins involved in a number of mitochondrial functions were found to be susceptible to modification by HNE, including components of the electron transport chain, the TCA cycle, as well as proteins involved amino acid metabolism and stress-responses. Implications of modification of these proteins by HNE are discussed. As HNE is produced in vivo during oxidative stress, the profile of mitochondrial targets of HNE was examined from Arabidopsis cell cultures exposed to various oxidative stress inducers. Menadione and hydrogen peroxide induced oxidative stress throughout the cell, while antimycin A initiated a mitochondrial targeted stress. A differential profile of mitochondrial proteins was observed to be modified by HNE in the various treatments. These results also showed that induction of stress within a whole cell can impact lipid peroxidation within the mitochondria. Overall, this work showed the presence and production of HNE in plant cells, and that HNE, both exogenous and endogenous, has the ability to modify a specific subset of mitochondrial proteins. In several cases this HNE modification was shown to have functional or structural consequences.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 5 uppsatser.

Thesis (M.Sc.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Plant Biology, Department of Biological Sciences. Title from pdf file main screen (viewed on April 23, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "August 2003." Typescript. Vita. Includes bibliographical references (leaves 158-174). Also issued on the Internet.

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.