The process of embryogenesis in higher plants is a critical stage of the sporophytic life cycle, transforming the fertilised egg cell via a precise sequence of events into a multi-cellular organism. It is during embryogenesis that the body plan of the developing plant is established. Analysis of transcriptional changesembryo. This thesis demonstrates the application of laser capture micro dissection to the analysis of embryogenesis in the model plant Arabidopsis thaliana. This technique has been used in combination with DNA microarray technology to allow a global analysis of gene expression in the cotyledon, root and shoot apical meristem regions of the torpedo-stage embryo. Validation of the approach has been achieved by comparison of the ATH1 GeneChip® data obtained, with published gene expression patterns confirmed by in situ hybridisation and promoter: GUS analysis. Further validation was successfully undertaken through the creation of promoter: Gus constructs for a number of previously uncharacterised putative transcription factor genes, selected on the basis of differential expression between the cotyledon and root regions. Initial attempts to assign putative function to these genes through an analysis of T-DNA insertion lines yielded no aberrant phenotypes. Transcriptional profiling of embryogenesis from the globular-stage through to the torpedo-stage was carried out using GeneSpring, uncovering distinct spatial and temporal expression patterns, and revealing a number of genes of potential interest for further research.

No description available.

The apoplastic enzyme, ascorbate oxidase (AO), is a blue copper oxidase that catalyses oxidation of ascorbate (AsA) to monodehydroascorbate (MDHA). In Arabidopsis thaliana, AO is encoded by three genes (At4g39830, At5g21105 and At5g21100) designated AO1, AO2, and AO3 respectively. Since AsA is the most abundant antioxidant in the apoplast and AO is active in this compartment, the regulation of apoplastic AsA redox status by AO and its role in development and environmental perturbations has become a subject of interest. Phylogenetic analysis showed that AO is present in higher plants, pteridophytes, mosses and green algae. Amino acid sequence analysis showed that AO2 and AO3 shared higher sequence identity than AO1. In silico analyses found that AO1 had a distinct expression pattern and subcellular localisation compared to AO2 and AO3, suggesting AO1 might be involved in alternative functions. Consistent with previous studies, AO activity was high in actively growing tissue of wild-type (WT) A. thaliana, supporting a possible role of AO in cell expansion. ao1, ao3 and ao1ao3 T-DNA insertion mutants were characterised. ao1 had similar level of AO activity to WT, while ao3 and ao1ao3 had 10-20% of WT AO activity. Compared with WT, these T-DNA insertion mutants did not show any phenotypic differences under unstressed or stressed (high light and drought) growth conditions. An artificial microRNA construct (amiR-AO) to silence all three AO genes was developed. Also, an overexpression plasmid (35S::AO3) harbouring AO3 gene was constructed. These constructs were used to transform A. thaliana. AO activity was undetectable in the amiR-AO line, while the 35S::AO3 line had 3-fold higher AO activity than the WT. Under unstressed normal growth conditions, the amiR-AO line had bigger rosette size, whereas the 35S::AO3 line exhibited early flowering and smaller number of rosette leaves. The amiR-AO line accumulated more anthocyanin and AsA than WT when acclimated to high light, whereas the 35S::AO3 line accumulated less anthocyanin than WT. In response to drought, the amiR-AO line did not show phenotypic differences compared to WT, while the 35::AO3 line had higher rate of leaf water loss and appeared to have greater sensitivity to drought. These results suggest that AO perturbation could, to some extent, affect the growth and stress response of A. thaliana although the effect is small.

Plant growth regulators have a vital role in plant growth and development. The cellular response to these regulators depends on the presence and the action of specific receptors. The plant growth regulators and their receptors act together in complexes which determine the final effects of the plant growth regulators. In the research reported here, emphasis has been given to the regulation of the activity of the receptors themselves. The regulation of the N-l-naphthylphthalamic acid (N~A) receptor through phosphorylation and dephosphorylation and the regulation of the auxin binding protein (ABP) through gene manipulation have been investigated. NPA, an auxin transport inhibitor, was found to bind specifically to a crude membrane preparation from sugar beet seedling leaf cell suspension cultures. The in vitro binding was optimal at pH 4.5 and 4?C. Binding parameters for NP A binding were determined by Scatchard analysis. The dissociation constant (Kd) and binding protein concentration were found to be 1.71 x 10-7 mol dm-3 and 220 pmoles g-I membrane protein respectively. It was found that the amount of specific 3H-NPA binding was significantly increased by adding Mg2+ A TP to the binding assay solution; treatment of membrane preparations with acid phosphatase, prior to the NP A binding assay, resulted in lower specific binding. A TP activation and phosphatase inactivation were culture stage dependent. Although a considerable effect could be detected when using cells from day 8 (representing the linear phase), the same treatment did not alter the binding if cells from day I (representing lag phase) or day 14 (representing the stationary phase) were used. These observations have strongly highlighted the possible involvement of a phosphorylation and dephosphorylation mechanism in vivo in the regulation of the activity of the NP A receptor. High phosphatase activity was found in the supernatant, but not in the membrane pellet, after 50,000 g centrifugation. The presence of a membrane-bound auxin receptor, ABP, was demonstrated by Scatchard analysis in sugar beet seedlings. The Kd value and the receptor concentration were found to be 2.15 x 10-6 mol dm-3 and 68 pmoles g-I membrane protein. The protein could be solubilised either with the detergent Triton X-I 00 or by acetone-washing, with a recovery of about 40%. An acetone-solubilised ABP preparation could be partially purified by DEAE-Sephacel ion exchange chromatography, NAA-linked AH-Sepharose 4B affmity chromatography or Sephadex G-200 gel filtration. The recovery after any of these chromatographic treatments was very low so that successive chromatography for further purification was unsuccessful. The low level of detectable binding after purification resulted mainly from the low abundance of ABP in the plant material. Non-radioactive labelling and detection techniques were used to show that an ABP-probe hybridized to sugar beet genomic DNA during dot blotting. The present study has indicated that receptor activity could be regulated by a phosphorylation and dephosphorylation mechanism in plants. The investigation has also suggested that the effect of plant growth regulators on plant development could be regulated through the manipulation of the expression of their receptor genes.

Les feuilles des plantes sont des organes importants pour la production de biomasse dans la nature car elles sont le siège principal de la photosynthèse, qui permet de transformer la matière minérale en matière organique. Identifier les mécanismes responsables de la morphogenèse, i.e. la genèse de la forme pendant le développement, est donc une question d'intérêt. Pour être analysée, la morphogenèse doit être appréhendée tout au long de la croissance car la forme finale d'une feuille est le résultat de mécanismes coordonnés dans l'espace et le temps. Pour comprendre ce type de processus complexes, la modélisation est une approche de choix. L'objectif de cette thèse était donc de développer des stratégies de quantification et de modélisation de la morphogenèse pour mieux comprendre le développement des feuilles. Pour quantifier la morphogenèse, ma première contribution a été de développer des méthodes pour dater précisément l'apparition des feuilles sur la plante et celle des dentelures sur la marge foliaire, ce qui permet de recaler dans le temps et comparer différentes feuilles en croissance. En calculant les trajectoires de croissance de feuilles moyennes, il est alors possible de préciser où et quand le développement de feuilles peuvent différer, au niveau global ou des dentelures, pendant la croissance. En analysant des feuilles de formes différentes de la plante modèle Arabidopsis thaliana, j'ai ainsi pu montrer que malgré des différences importantes en taille et forme globale, il y a une similarité dans le développement des dentelures. Ces résultats suggèrent qu'il existe des processus identiques qui gouvernent l'apparition et la croissance des dentelures. J'ai ensuite proposé un modèle de développement des feuilles, à partir duquel il est possible de simuler la croissance d'une feuille. Il est basé sur des mécanismes biologiques qui on été identifiés comme étant importants dans la mise en place de la forme. Pour paramétrer le modèle, une approche d'optimisation a été mise au point pour déterminer les paramètres optimaux du modèle. Les résultats obtenus montrent que l'apparition séquentielle des dents ainsi que certains paramètres morphologiques peuvent être bien reproduits par le modèle. / Plant leaves are important for the production of biomass in nature, because they are the main site of photosynthesis, They have various shapes and it has been shown that their morphology influences photosynthesis efficiency. Identifying the mechanisms responsible for morphogenesis, i.e. the genesis of the shape during development, is therefore a matter of interest. To be analyzed, morphogenesis must be apprehended throughout the whole growth because the leaf final form is the result of coordinated mechanisms in space and time. To understand this type of complex processes, modeling is an approach of choice. Consequently, the objective of this thesis was to develop strategies for the quantification and modeling of morphogenesis to better understand leaf development. To quantify morphogenesis, my first contribution was to develop methods to precisely date the appearance of the leaves on the plant, and of the serrations at the leaf margin, allowing to register in time and to compare different growing leaves. Besides, based on mean growth trajectories, it is possible to specify where and when the developments of different leaves differ, at global and serration scales, during growth.By analyzing the development of leaves of the plant model Arabidopsis thaliana that have different shapes, in wild type or in mutants, it has been shown that, despite significant differences in leaf size and shape, there is a similarity in the development of all serrations. These results suggest that there are identical processes that control the appearance and growth of serrations. I proposed two leaf development models, based on biological mechanisms that have been identified, in the literature, as important for the leaf shaping, and also on the quantification of leaf morphogenesis performed in this work. The simulation module, that generates growth trajectories from the model, makes it possible to compare simulated and real developments. To parameterize the model, an optimization approach has been proposed to determine optimal parameters, which minimizes the differences between simulation and real growths. The results showed that the sequential appearance of the teeth as well as important morphological characteristics can be well reproduced by the models.

B. Cinerea, l'agent responsable de la pourriture grise, possède deux alpha-tubulines : l'alpha-tubuline A et l'alpha-tubuline B. Ces protéines sont connues pour participer à la construction des microtubules, mais la connaissance de leurs rôles est relativement limitée chez les champignons et particulièrement chez les pathogènes des plantes. L'analyse du profil d'expression des gènes codant pour ces tubulines a démontré que l'alpha-tubuline B est majoritairement exprimé et qu'il se caractérise par un pic au moment de la germination de la spore fongique. L'étude du promoteur de son gène a révélé la présence de trois zones possibles de régulation, et l'approche de «simple-hybride» a permis de mettre en évidence deux régulateurs potentiels : BcYOH1 et BcFT027. Un mini-réseau de régulation entre les différents gènes codants pour les tubulines et ces facteurs de transcription a été mis en évidence. Nos résultats ont montré que le gène codant pour l'alpha-tubuline B n'est pas indispensable à la vie cellulaire et l'étude du mutant de délétion a permis d'attribuer un rôle particulier de cette protéine dans la formation du mycélium aérien et, donc dans la production des spores de la reproduction asexuée, mais également dans le cycle infectieux du champignon. Par ailleurs, les mutants de délétion des gènes codant pour les deux facteurs de transcription identifiés dans notre étude ont permis l'analyse de leur rôle sur le développement et le pouvoir infectieux du champignon. Nous avons ainsi montré que la vitesse de croissance, la biomasse, le mycélium aérien et la vitesse d'infection étaient augmentés chez le mutant BcFT027 / The plant pathogen Botrytis cinerea contains two alpha-tubulin isomers (alpha-tubulin A and B). These proteins are known to participate in the construction of microtubules in all eukaryotes, but our knowledge about the roles of different isomers is particularly poor in fungi, and null in fungal plant pathogens. Analysis of gene expression profiles in B. cinerea revealed that the alpha-tubulin B encoding gene is more expressed than the alpha-tubulin A one and that its expression peaks during spore germination. Subsequent promoter studies led to the identification of three DNA regions probably involved in this regulation, and two putative regulators were then found by using the one-hybrid yeast system : the already discovered BcYOH1 and the totally unknown and ascomycetous specific BcFT027. Additional expression studies in mutant strains of these regulators finally suggested the existence of a regulatory network between these two regulators and the two alpha-tubulin encoding genes. Production and analysis of alpha-tubulin B deletion mutants showed that this isomer is not essential for cell viability in B. cinerea. More importantly, this study revealed that the alpha-tubulin protein plays a role during plant infection as well as in the formation of aerial mycelium and the production of asexual spores. Partial to complete characterization of the BcYOH1 and BcFT027 deletion mutants strengthened these results and showed that BcFT027 is a key player in the development of areal mycelium and of the infection process (via the development of penetration structures called infection cushions). Never reported before, these results are of significant interest in our understanding of tubulins and fungal development

Les populations naturelles sont confrontées à des changements environnementaux. Pour y faire face, différentes réponses ont été sélectionnées au cours de l'évolution. Parmi elles se trouvent la plasticité phénotypique et l'adaptation génétique. Etudier les liens existants entre elles est une manière de comprendre les dynamiques des populations et de prévoir leurs réponses à un environnement changeant. Dans la présente étude, je me suis attaché à étudier ces liens à plusieurs échelles (intra- et interspécifique), chez le complexe d'espèces cryptiques de la micro-algue Alexandrium minutum, et ce à la fois in vitro et in situ. En ce qui concerne la plasticité phénotypique, ces deux espèces proches montrent de profondes différences, soulignant les liens entre divergence génétique et écologique. Au niveau intraspécifique, il apparaît que face à des variations de facteurs abiotiques, les populations ajustent les niveaux d'expression de certains gènes (notamment impliqués dans des fonctions de motilité et d'interactions intercellulaires dans des environnements froids à faible salinité). D'autre part, les populations montrent de la différentiation génétique à la fois à faible échelle spatiale, au cours du temps, et lorsque la communauté change. Pour conclure, il existe une interaction directe entre divergence génétique et changements d'expression de gènes. En plus de poser de nombreuses questions quant aux capacités de réponse des populations, ces résultats soulignent comment plasticité phénotypique et changements génétique sont liés et interagissent. Ils offrent une perspective nouvelle sur les mécanismes qui sous-tendent les réponses des populations à leur environnement. / Natural populations face environmental changes. In this context, different responses were evolutionnary selected. Among them are phenotypic plasticity and genetic adaptation. Studying the links between these two types of response is a way to understand population dynamics and to predict how they may respond to a changing environment. In the present Ph.D thesis, I focused on studying these links at several scales (intra- and interspecific), in the cryptic species complex of the microalga Alexandrium minutum, both in vitro and in situ. With respect to phenotypic plasticity, these two closely related species show profound differences, highlighting the links between genetic and ecological divergence. At the intraspecific level, it appears that, when facing abiotic factors variations, populations adjust the expression levels of certain genes (notably involved in motility related functions and intercellular interactions under low-salinity and cold environments). On the other hand, populations show genetic differentiation at both small spatial scale, over time, and when the community changes. To conclude, there is a direct interaction between genetic divergence and changes in gene expression. In addition to asking many questions about the response capabilities of populations, these results highlight how phenotypic plasticity and genetic changes are linked and interact. They offer new perspectives on the mechanisms underlying population responses to their environment.

Malgré leur importance dans le déterminisme du sexe chez de nombreux organismes, les chromosomes sexuels ont été étudiés chez quelques espèces seulement du fait du manque de séquences disponibles. En effet, le séquençage et l'assemblage des chromosomes sexuels est rendu très difficile par leurs abondantes séquences répétées. Durant cette thèse, une méthode probabiliste a été développée pour inférer les gènes liés au sexe à partir de données RNA-seq chez une famille. Des tests de cette méthode appelée SEX-DETector sur des données réelles et simulées suggèrent qu'elle fonctionnera sur une grande variété de systèmes. La méthode a inféré ∼1300 gènes liés au sexe chez Silene latifolia, une plante dioïque qui possède des chromosomes sexuels XY pour lesquels quelques données de séquence sont disponibles (dont certaines obtenues lors de cette thèse par séquençage de BACs). Les gènes du Y sont moins exprimés que ceux du X chez S. latifolia, mais le statut de la compensation de dosage (un mécanisme qui corrige la sous-expression des gènes liés au sexe chez les males) est encore controversé. L'analyse des nouveaux gènes liés au sexe inférés par SEX-DETector a permis de confirmer la compensation de dosage chez S. latifolia, qui est effectuée par la surexpression du X maternel, possiblement via un mécanisme epigénétique d'empreinte. Les données ont également été utilisées pour étudier l'évolution de l'expression biaisée pour le sexe chez S. latifolia et ont révélé que la majorité des changements de niveaux d'expression ont eu lieu chez les femelles. Les implications de nos résultats concernant l'évolution de la dioécie et des chromosomes sexuels sont discutés / In many organisms, sexes are determined by sex chromosomes. However, studies have been greatly limited by the paucity of sex chromosome sequences. Indeed, sequencing and assembling sex chromosomes are very challenging due to the large quantity of repetitive DNA that these chromosomes comprise. In this PhD, a probabilistic method was developed to infer sex-linked genes from RNA-seq data of a family (parents and progeny of each sex). The method, called SEX-DETector, was tested on simulated and real data and should performwell on a wide variety of sex chomosome systems. This new method was applied to Silene latifolia, a dioecious plant with XY system, for which partial sequence data on sex chromosomes are available (some of which obtained during this PhD by BAC sequencing), SEX-DETector returned ∼1300 sex-linked genes. In S. latifolia, Y genes are less expressed than their X counterparts. Dosage compensation (a mechanism that corrects for reduced dosage due to Y degeneration in males) was previously tested in S. latifolia, but different studies returned conflicting results. The analysis of the new set of sex-linked genes confirmed the existence of dosage compensation in S. latifolia, which seems to be achieved by the hyperexpression of the maternal X chromosome in males. An imprinting mechanism might underlie dosage compensation in that species. The RNAseq datawere also used to study the evolution of differential expression among sexes in S. latifolia, and revealed that in this species most changes have affected the female sex. The implications of our results for the evolution of dioecy and sex chromosomes in plants are discussed