La polyploïdie consiste en la duplication du génome entier et constitue une force évolutive majeure chez les eucaryotes, notamment chez les angiospermes. Les espèces du genre Brachypodium ont émergé comme une modèle intéressant des monocotylédones. Parmi ces espèces, l’allopolyploïde B. hybridum (2n = 30) a résulté de l’hybridation entre B. distachyon (2n = 10) et B. stacei (2n = 20). Les deux espèces parentales ont eu des évolutions chromosomiques assez divergentes aboutissant à ce que B. distachyon possède deux fois moins de chromosomes qui sont néanmoins deux fois plus grands que ceux de B. stacei.L'objectif de ma thèse est de développer un modèle de Brachypodium polyploïde en synthétisant des autopolyploïdes et des allopolyploïdes ainsi que les caractérisant aux niveaux phénotypique, cytogénétique et génomique.Les autotétraploïdes ont été générées par traitement à la cochicine de deux lignées de B. distachyon et trois autres de B. stacei. Tous les autopolyploïdes obtenues ont été validés par cytométrtie de flux et ont montré une stabilité de caryotype et de phénotype, exceptés ceux de la lignée ABR114 de B. stacei qui ont montré des descendances autotetraploides et aneuploïdes. Les comparaisons des caractères de l’inflorescence et des ligules montrent que les autotétraploïdes des deux espèces dépassent généralement leurs progéniteurs diploïdes, mais une faible fertilité comme illustré par le faible nombre de graines par inflorescence.Les allotétraploïdes synthétiques, similaires à B. hybridum, ont été générés par hybridation interspécifique entre différentes lignées de B. distachyon et B. stacei. Alors que les hybrides interspécifiques F1 stériles ont été obtenus lorsque B. distachyon a été utilisée comme parent maternel (0,15% ou 0,245% des croisements réalisés), aucun hybride a été obtenu à partir des croisements réciproques ou lorsque des plantes autotetraploïdes des espèces parentales ont été croisées. Le doublement du génome par traitement à la colchicine a restauré la fertilité et les plantes F1 doublées produisent par auto-polllinisation quelques graines S1 (première génération autofécondation). Les plantes S1 de la l’allo3-1×5 étaient fertiles donnant des générations suivantes alors que ceux de l’allo21×114 étaient stériles. Ces allotétraploïdes se sont montrées stables au niveau phenotypique, cytogénétique et génomiques, ressemblant ainsi le B. hybridum naturel.J’ai ainsi utilisé la méthodologie Illumina de nouvelles générations de séquençage pour caractériser ces différents polyploïdes aux niveaux: (i) Des variations structurales par reséquençage de l’ADN, (ii) Le transcriptome par l'ARN-Seq afin de caractériser les modifications de l’expression des gènes (iii) la méthylation CpG par séquençage de l’ADN traité au bisulfite. Une large gamme de possibilités de comparaison pourrait être réalisée lorsque les séquences des génomes des trois espèces naturelles de Brachypodium seraient disponibles.La disponibilité de la séquence du génome de B. distachyon a permis de réaliser une analyse pilote comparant l’expression des gènes dupliqués dans les autopolyploïdes de la lignée Bd3-1 à ceux des diploïdes. Une expression similaire pour 95,25% des gènes a été observée et seulement 4,75% des gènes ont montrés une expression différentielle. La majeure partie de ces derniers (1053 gènes, 82%) étaient moins exprimés dans les autopolyploides. Des fonctions géniques tel que la ségrégation des chromosomes nucléaires, la réparation de l'ADN, la recombinaison homologue, la régulation de la transcription se sont révélées enrichies.La création des autopolyploides et allopolyploides et leurs caractérisations avec les outils NGS offrent la possibilité d’investir de façon intégrée les secrets de succès des polyploïdes. / Polyploidy consisting in whole genome duplication is an important evolutionary force widespread in eukaryote and very prominent in angiosperms. Species of the plant Brachypodium genus emerged as an important monocot and polyploid model. Among these species, the annual allopolyploid B. hybridum (2n=30), derived from hybridizations between B. distachyon (2n=10) and B. stacei (2n=20), was shown to be polyphyletic. The two parental species have similar genome content and ploidy level but an asymmetric chromosome evolution where B. distachyon has two times less chromosomes that are two times bigger than those of B. stacei.The objective of my PhD program consisted in developing a valuable Brachypodium polyploid model by synthesizing autopolyploids and allopolyploids and then their characterizing at the phenotype, cytogenetic and genomic levels.Autotetraploids were generated from two inbred lines of B. distachyon and three ones of B. stacei, through colchicine treatments. The genome doubling was validated by flow cytometry and karyotyping with fluorescent in situ hybridization analyses. All autopolyploids showed stability in phenotype as well as karyotype except those of line ABR114 of B. stacei that showed various aneuploid progenies. Quantitative comparison of inflorescences and flag leaves characters showed that both B. distachyon and B. stacei autotetraploids generally exceeded their diploid progenitors, but their fertility was reduced as illustrated by the lower number of seeds per inflorescence.Synthetic allotetraploids were generated through interspecific hybridization between various lines of B. distachyon and B. stacei species. While sterile amphihaploid F1 interspecific hybrids were obtained at low frequencies (0,15% or 0,245% of crosses) when B. distachyon was used as the maternal parent, no hybrids were obtained from reciprocal crosses or when autotetraploids of the parental species were crossed. Genome doubling through colchicine treatment restored fertility where doubled F1 plants produced a few S1 seeds (first selfed generation). S1 plants of allo3-1×5 were fertile and gave rise to further generations whereas those of allo21×114 were sterile. The synthetic allotetraploids were shown to be highly-stable and resembled the natural B. hybridum allopolyploid at the phenotypic, cytogenetic and genomic levels.I have used the Illumina next generation sequencing (NGS) methodology to characterize the different polyploids at the levels of: (i) The DNA resequencing to reveal genetic changes, (ii) The transcriptome analysis through RNA-Sequencing and (iii) CpG methylation through bisulfite sequencing. A total of 13 genotypes containing synthetic autopolyploids, synthetic allopolyploids and the natural diploid species B. distachyon, B. stacei and the allopolyploid B. hybridum have characterized. A wide range of possibilities of comparison could be then realized, when the genomes sequences of all three species will be available.The availability of sequence genome sequence of B. distachyon allowed a pilot gene expression comparison between diploids and autopolyploids of Bd3-1. Diploids and autotetrapolyploids of of Bd3-1 showed similar expression for most of the genes (95.25%). Only 4.75% of total genes were differentially expressed genes, the major proportion of which (1053 genes, 82%) were down-expressed in autotetraploids with important enrichment in genome maintenance functions such as nuclear chromosome segregation, DNA repair, DNA replication, homologous recombination.The successful creation of stable autotetraploids and synthetic B. hybridum allopolyploids together with genome wide characterization using NGS offer the possibility to unravel clues of success of polyploidy in angiosperms.

La symbiose endomycorhizienne à arbuscules (SMA) a une importance écologique et agronomique majeure car elle permet à la majorité des plantes terrestres, via une association avec des champignons du phylum des Glomeromycota, une meilleure acquisition de nutriments du sol. Les champignons grâce à un large réseau mycélien dans le sol, collectent et echangent des nutriments avec les plantes qui leurs fournissent des substrats carbonés issus de la photosynthèse. Le champignon mycorhizienne à arbuscules (CMA) Rhizophagus irregularis, capable de coloniser les racines de la plupart des plantes terrestres, sécrète des lipo-chitooligosaccharides (Myc-LCOs) et des chitooligosaccharides à chaîne courte (Myc-COs). L’ajout exogène de Myc-LCO stimule la colonisation de plusieurs espèces par des CMAs. Ces Myc-LCOs et les Myc-COs sont capables d’activer une voie de signalisation symbiotique requise pour l’établissement de la SMA. Cependant, le rôle de ces molécules et leur importance dans l’établissement de la symbiose restent à ce jour inconnu. Au cours de ma thèsej’ao étudié la SMA chez la plante modèle pour les monocotylédones tempérées, Brachypodium distachyon. Je me suis intéressé, tout d’abord, à la perception des signaux fongiques (Myc-LCOs et Myc-COs). Un mécanisme possible par lequel les Myc-LCOs pourraient stimuler la SMA, est leur capacité à induire une augmentation du nombre de racines latérales (RL), montrée chez la légumineuse modèle Medicago truncatula ainsi que chez la monocotylédone Oriza sativa (riz). Les réponses transcriptionnelles aux Myc-LCOs chez M. truncatula suggèrent une interaction entre la perception de Myc-LCOs et la signalisation auxine. Par ailleurs, l’ajout d’auxine stimule la SMA chez M. truncatula et le riz. Pendant mes travaux de thèse, nous avons pu montrer que des LCOs induisent aussi la formation de RLs chez B. distachyon. De plus, nous avons pu montrer un effet des LCOs sur l’homéostasie de l’auxine. Finalement, nous avons pu confirmer un rôle positif de l’auxine dans la mise en place de la SMA grâce à l’étude d’un mutant surproduisant de l’auxine. Par ailleurs, des travaux dans l’équipe ont permis d’identifier BdLYR1, un récepteur kinase contenant des motifs lysin (LysM-RLK) capable de lier les LCOs à haute affinité. Cependant, un mutant dans ce gène ne présente aucun phénotype mycorhizien suggérant une redondance fonctionnelle au niveau des récepteurs et/ou des signaux pour l’activation de la voie de signalisation symbiotique. J’ai initié la caractérisation du LysM-RLK BdLYR4, un récepteur potentiel de COs. Enfin, il a été observé que la réponse de croissance des plantes à la colonisation par les CMA est à la fois dépendantes de l’environnement, de la souche de champignon utilisée ainsi que du génotype de plante. Très peu d’études ont été réalisées dans le but d’identifier les mécanismes moléculaires qui régissent ces réponses de croissance. Pour pouvoir le faire, j’ai initié la caractérisation de la variabilité génétique pour les réponses de croissance aux CMAs chez B. distachyon dans le but d’identifier des génotypes contrastés ainsi que les variables phénotypiques les plus pertinentes à analyser. Ces travaux ont permis de trouver des conditions de culture qui permettent l’analyse de la réponse croissance chez B. distachyon. / The endomycorrhizal symbiosis with arbuscules (AM) is of major ecological and agronomic importance because it allows the majority of terrestrial plants, via an association with phylum fungi of Glomeromycota, to facilitate their acquisition of nutrients and water. Fungi through a large soil exchange surface collect and provide nutrients to plants that provide fungi with carbonaceous substrates derived from photosynthesis. The fungus AM Rhizophagus irregularis, capable of colonizing the roots of most terrestrial plants, secretes lipo-chitooligosaccharides (Myc-LCOs) and short-chain chitooligosaccharides (Myc-COs). These molecules are capable of activating a signaling pathway required for the establishment of the AM symbiosis. However, their role and relevance for AM symbiosis is completely unkown. During my phD, I studied perception of fungal signals (Myc-LCOs and Myc-COs) in B.distachyon. Myc-LCOs stimulate AM symbiosis in several plants and induce an increase in the number of lateral roots (RL) in legume plant Medicago truncatula, as well as in Oriza sativa (rice).Transcriptional responses to LCO in M.truncatula suggest a crosstalk between auxin and LCO. Moreover, the addition of auxin also stimulates AM symbiosis in M. truncatula and rice. During my phD, we showed that LCOs stimulates also lateral root formation in B.distachyon. In addition, we showed that these developmental effects are linked to a regulation of auxin biosyntheses and homeostasis. Finally, I could confirm the positive role of auxin in AM thanks to an auxin-overproducer mutant. In the team, BdLYR1, a high-affinity LCO binding protein has been identified. However, the mutant Bdlyr1 is not affected in mycorrhization, as it was also shown for its ortholog in rice. This can be explained by functional redundancy of the receptors but also by redundancy of fungal signals (Myc-CO). During my phD, I focused on BdLYR4, which is a good candidate for Myc-COs receptor. It has been reported mycorrhizal growth responses (MGR) in colonized plants. These growth responses depend on environmental conditions, fungal strain and plant genotype. To date, very few studies have been done in order to identify molecular mechanisms controlling MGRs. To do so, I initiate the characterization of natural variability for MGRs in B.distachyon in order to identify contrasted genotypes and the more convenient phenotipical and physiological traits to analyze. All this work leads us to identify good conditions that maximize differences between genotypes.

It is common knowledge that invasive species cause worldwide ecological and economic damage, and are nearly impossible to eradicate. However, upon introduction to a novel environment, alien species should be the underdogs: They are present in small numbers, possess low genetic diversity, and have not adapted to the climate and competitors present in the new habitat. So, how are alien species able to invade an environment occupied by native species that have already adapted to the local environment? To discover some answers to this apparent paradox I conducted four ecological genetic studies that utilized the invasive species Brachypodium sylvaticum (Hudson) Beauv. to determine mechanisms contributing to adaptation and success in the novel habitat. The first study used simulations and experiments to test the hypothesis that genetic purging, the process where genetic load is reduced by selection against the recessive deleterious alleles expressed in the homozygous state, promotes invasive range expansion. I found that homozygous populations on B. sylvaticum's range periphery displayed lower inbreeding depression compared to heterozygous populations near introduction sites. Empirical tests with B. sylvaticum further demonstrate that purging of genetic load is a plausible scenario promoting range expansion during invasion. Next, I explored how the interaction between population genetic diversity and the environment contributed to the establishment and spread of Brachypodium sylvaticum. I found that nitrogen application increases both final size and shoot biomass for B. sylvaticum individuals from source populations with low HS levels to levels found in individuals from populations with high HS. A coefficient of relative competition intensity index (RCI) displayed reduced effects of interspecific competition on B. sylvaticum biomass in high nitrogen plots. Results show that elevated nitrogen deposition is a factor that increases establishment of introduced species with historically small effective population sizes. Thirdly, I investigated phenotypic differentiation during the establishment and range expansion of Brachypodium sylvaticum. Utilizing a novel approach, unique alleles were used to determine the genetic probability of contribution from native source regions to invasive regions. These probabilities were integrated into QST-FST comparisons to determine the influence of selection and genetic drift on twelve physiological and anatomical traits associated with drought stress. Phenotypic divergence greater than neutral expectations was found for five traits between native and invasive populations, indicating selective divergence. Results from this study show that the majority of divergence in B. sylvaticum occurred after introduction to the novel environment, but prior to invasive range expansion. The final chapter of my dissertation investigates the adaptive role of genetic differentiation and plasticity for Brachypodium sylvaticum invasion. Plasticity was measured across treatments of contrasting water availability. Linear and nonlinear selection gradients determined the effect of directional and quadratic selection on plasticity and genetic differentiation. Invasive trait divergence was a consequence of post-introduction selection leading to genetic differentiation, as there were no plastic responses to contrasting water availability for any measured traits. Genetic divergence of invasive plants was not consistently in the direction indicated by selection, suggesting limitations of selection that may be a consequence of physical constraints and/or tradeoffs between growth and abiotic tolerance. Results suggest that selection, rather than plasticity, is driving phenotypic change in the invaded environment. The combined volume of these studies contributes significantly to the field of invasion and plant biology by providing novel insights into the processes underlying range expansion, adaptation, and ultimately, evolution of introduced species.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-04-29T16:31:02Z No. of bitstreams: 1 texto completo.pdf: 3735449 bytes, checksum: aa20d55ef3b80cac2cc56637e94639c9 (MD5) / Made available in DSpace on 2016-04-29T16:31:02Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3735449 bytes, checksum: aa20d55ef3b80cac2cc56637e94639c9 (MD5) Previous issue date: 2013-10-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Brachypodium distachyon (L.) P. Beauv. (Poaceae: Poideae) tem se destacado como planta modelo para gramíneas de clima temperado e espécies usadas para a produção de biocombustíveis. A linhagem Bd21 de Brachypodium distachyon tem um genoma completamente seqüenciado e montado, além de protocolos de genômica e de transformação bem estabelecidos com base em calos embriogênicos. No entanto, as informações sobre a origem e as alterações celulares que ocorrem durante a diferenciação de embriões somáticos nos estágios iniciais não foi documentada para B. distachyon. Também não há relatos sobre o uso de abordagens moleculares para investigar o processo de embriogênese somática nesta espécie. Portanto, os objetivos deste trabalho foram: (1) caracterizar as alterações morfológicas, anatômicas e histoquímicas que ocorrem durante a indução de embriogênese somática a partir de embriões zigóticos imaturos (EZI) da B. distachyon linhagem de referência Bd21 usando microscopia de luz e de varredura em associação com testes histoquímicos e, (2), realizar a clonagem e caracterização de genes SERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE) e analisar sua expressão na indução de embriogênese somática utilizando hibridização in situ para monitorar os processos morfogenéticos in vitro. Culturas embriogênicas de B. distachyon (BD21) foram estabelecidas usando EZI 15 dias após a antese, em meio Murashige e Skoog (1962) contendo ácido 2,4- diclorofenoxiacético. A regeneração in vitro de plântulas derivadas de embriões somáticos ocorreu pela embriogênese somática através da via indireta. Os embriões somáticos tiveram uma origem multicelular e originaram-se de calo embriogênico formado a partir de células da epiderme na região do nó escutelar que se estenderam para a periferia do EZI. A ordem de acumulação de reservas nos embriões somáticos foi semelhante a dos embriões zigóticos. Nas culturas embriogênicas, proteínas e lipídios foram utilizados nos primeiros 2 dias em meio de indução. O teor de amido aumentou nos primeiros 2 dias em meio de indução e diminuiu em seguida em número de grânulos que se tornaram maiores e apareceram principalmente nas células vacuoladas adjacentes às massas pró-embriogênicas. Pequenos grânulos de amido começaram a acumular nos pró-embrioides após 4 dias em meio de indução e tornaram- se maior e mais abundantes em células do escutelo aos 12 dias em meio de indução. A diferenciação do embrião somático seguiu a mesma sequência de desenvolvimento verificado em outros membros da família Poaceae, ou seja, a passagem pelos estádios globular, escutelar e coleoptilar. O gene SERK tem sido usado como um marcador para as células competentes na embriogênese somática de várias espécies. Neste estudo, utilizando iniciadores degenerados, uma sequência específica homóloga de um fragmento do gene SERK foi amplificada de B. distachyon Bd21. A análise da sequência do fragmento de SERK (766 bp) revelou altos níveis de similaridade com genes SERK relatados em outras espécies. A análise de hibridização in situ mostrou que o gene SERK estava presente nos tecidos embriogênicos de B. distachyon antes do desenvolvimento de embriões somáticos e continuou sendo expresso nos estágios globular e escutelar. Estes resultados sugerem que a expressão do gene SERK de B. distachyon pode estar associada com a indução da embriogênese somática. Este estudo faz a primeira descrição de mudanças morfoanatômicas e histoquímicas durante o processo de embriogênese somática em B. distachyon linhagem Bd21. Este é também o primeiro relato sobre a clonagem e expressão de um gene SERK para a espécie e sugere que este gene pode servir como um marcador molecular para monitorar a transição de células somáticas em células competentes e embriogênicas também em B. distachyon. / Brachypodium distachyon (L.) P. Beauv. (Poaceae: Poideae) has been proposed as a new model for temperate and biofuel grasses. Brachypodium distachyon inbred line Bd21 has a fully sequenced and assembled genome, a series of genomics resources, and well-established somatic embryogenesis-based transformation protocols. However, information about origin and cellular changes occurring during the early differentiation of somatic embryos has not been documented for B. distachyon. There are also no reports on the use of molecular approaches to investigate somatic embryogenesis in B. distachyon. Therefore, the objectives of this study were (1) to characterize the morphological, anatomical and histochemical changes occurring during the induction of somatic embryogenesis from immature zygotic embryos (IZE) of B. distachyon community standard line Bd21 using light and scanning electron microscopy in association with histochemical tests and, (2), to carry out the cloning and characterization of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes and analysis of its expression in somatic embryogenesis induction using in situ hybridization for monitoring the morphogenetic processes in vitro. Somatic embryogenic cultures of B. distachyon (Bd21) were established following culture of IZE, 15 days post anthesis, on Murashige and Skoog (1962) medium containing 2,4- dichlorophenoxyacetic acid. In vitro regeneration of plantlets derived from somatic embryos occurred by the indirect somatic embryogenesis pathway. Somatic embryos had a multicellular origin and originated from embryogenic callus formed from cells of the epidermis in the region of the scutellar node and extended to the periphery of IZE. The order in accumulation of storage reserves in somatic embryos was similar to that of zygotic embryos. In the embryogenic cultures, storage proteins and lipids were used up in the first 2 days after culture (DAC). The levels of starch increased in the first 2 DAC and then decreased in number of granules that became larger and appeared mainly in the vacuolated cells subtending the proembryonic masses. Small starch granules started accumulating in proembryoids after 4 DAC and became larger and abundant in scutellar cells 12 DAC. Somatic embryo differentiation in B. distachyon proceeded through globular, scutellar and coleoptilar stages, following the morphological pattern of development of that reported in other members of the Poaceae. The SERK gene has been used successfully as a marker for competent cells in somatic embryogenesis of several species. In this study, using degenerate primers, it was possible to amplify a specific homologous sequence of a SERK gene fragment from B. distachyon Bd21. Sequence analysis of the SERK fragment (766 bp) revealed high levels of similarity to other reported SERKs. In situ hybridization analysis showed that the SERK gene was present in embryogenic tissues of B. distachyon before somatic embryo development and continued expressing through globular and scutellar stages. These results suggest that the expression of the B. distachyon SERK gene was associated with induction of somatic embryogenesis. This study provides the first description of morphoanatomical and histochemical changes underlying the embryogenic process in B. distachyon reference line Bd21. It is also the first report on the cloning and expression of a SERK gene for the species, suggesting that it could serve as a molecular marker to monitor the transition of IZE cells into competent and embryogenic cells also in B. distachyon line Bd21.

La production de biocarburants de deuxième génération basée sur la transformation de la biomasse végétale est une question d’actualité. La biomasse végétale est représentée par les parois des cellules, qui consistent en un réseau de microfibrilles de cellulose et de polysaccharides enchâssés dans de la lignine. Pour exploiter pleinement le potentiel de cette biomasse, il est nécessaire d’apporter des connaissances complémentaires sur les mécanismes de biosynthèse de ces polymères pariétaux. Par exemple, il est important d’améliorer le rendement de saccharification des microfibrilles de cellulose afin de produire de plus grandes quantités de bioéthanol. Nous avons donc combiné des études basées sur le modèle bien connu Arabidopsis et sur Brachypodium distachyon, la nouvelle espèce modèle pour les graminées tempérées et les céréales monocotylédones dédiées à la production de biocarburants. La cellulose est synthétisée par des complexes membranaires de cellulose synthases (CSC) qui contiennent les sous-unités catalytiques de cellulose synthase (CESAs), et cela requiert d’autres partenaires parmi lesquels KOR1, une endo-β-1,4-glucanase. Le trafic intracellulaire des CESAs semble jouer un rôle crucial dans la régulation du niveau de la synthèse de la cellulose. Nous avons étudié en détails le trafic intracellulaire de KOR1 dans des hypocotyles d’Arabidopsis cultivés à l’obscurité. En parallèle, lors d’un crible visuel de la collection de mutants de Brachypodium de l’INRA de Versailles, nous avons sélectionné un mutant nommé spa. Ce mutant partage des caractéristiques avec les mutants brittle culm du riz et de l’orge, comme par exemple des tiges cassantes, un xylème irrégulier, et une importante déficience en cellulose, surtout au niveau des tiges, qui contiennent 50% de la quantité retrouvée dans une plante sauvage. Des dosages de lignine ont montré une augmentation significative chez spa. De façon itnéressante, ce mutant présente un défaut flagrant du port érigé, au contraire des mutants brittle culm qui sont parfaitement érigés. Les défauts mécaniques du mutant spa s’illustrent par un module de Young trois fois inférieur à celui d’une plante sauvage. Des approches complémentaires ont été mise en œuvre afin d’identifier les défauts génétiques responsables de ce phénotype : le séquençage de gènes candidats reliés à la synthèse de la cellulose a été réalisé ainsi qu’une approche de NGS. De plus, dans le cadre du projet Européen RENEWALL et du projet KBBE CellWall, et grâce à l’outil BradiNet (M.Mutvil, KBBE CellWall) permettant d’accéder aux réseaux de co-expression, des stratégies RNAi sont en cours afin d’inactiver certains gènes seléctionnés selon des critères d’expression spécifiques, et selon leur implication potentielle dans la synthèse de la cellulose, spécifiquement chez les monocotylédones. Parmi ces gènes nous nous sommes concentrés sur la famille des MAP65 (Microtubules Associated Proteins), qui pourraient, au vu de la relation étroite entre microtubules et microfibrilles, jouer un rôle dans la déposition de la cellulose. / The production of second-generation biofuels based on the transformation of plant biomass is a pressing issue. Biomass is represented by cell walls of the plant cells consisting of a network of cellulose microfibrils and polysaccharides encrusted by lignin. To enhance the potential of plant biomass, we need to provide insights on the mechanisms of the biosynthesis of cell wall polymers. For example, it is important to improve the saccharification yield of cellulose microfibrils to produce the highest amount of bioethanol. We therefore combine studies on the well-known model plant Arabidopsis and Brachypodium distachyon, the new model species for temperate graminae and monocotyledonous crops dedicated to biofuel production. Cellulose is synthesized by plasma membrane-bound cellulose synthase complexes (CSC) containing cellulose synthase proteins (CESAs) and requires other partners among which the endo-beta1,4 glucanase KOR1. The intracellular trafficking of CESAs seems to be crucial to regulate the cellulose synthesis rate. We investigated in detail the intracellular trafficking of KOR1 in Arabidospis dark-grown hypocotyls.In parallel we selected by visual screening of the Versailles collection of mutagenized Brachypodium distachyon a mutant called spa. This mutant shares characteristics of the brittle culm mutants of rice and barley, such as brittleness, irregular xylem, and a cellulose content deficiency especially in stems, with 50% of the amount found in the wild type. Lignin assays indicate a higher amount of lignin in spa. Interestingly, this mutant is also "floppy" unlike others brittle culm mutants which are fully erected and the mechanical strength defects of spa is illustrated by a Young’s modulus three times lower than that of WT. Complementary approaches were used to identify the SPA gene: sequencing of candidate genes related to cell wall synthesis or co-expressed with secondary cell wall cellulose synthases and a classical mapping strategy combined with NGS methods. Moreover within the framework of the European RENEWALL and KBBE CellWall projects and thanks to the co-expression network tool BradiNet (M. Mutwil, KBBE project), RNAi strategies are in progress to inactivate a few genes selected according to specific expression criteria and potentially involved in cell wall synthesis specifically in monocots. Among these genes we are focusing on the MAP65 family (Microtubules Associated Proteins), which could play a role in cellulose deposition according to the close relationship between microfibrils and microtubules.

Evolution of genotypes during range expansion is driven in part by colonization dynamics. I investigated genetic patterns of colonization and dispersal during initial expansion of an invasive bunchgrass, Brachypodium sylvaticum, into Oregon. Using microsatellite markers, I sampled plants at two different scales: at regular intervals along three parallel roads spanning about 30km, and in populations identified throughout Oregon. I also collected field-generated progeny from a subset of populations and used molecular identification of outcrossing events to estimate selfing rates in both central and peripheral populations. Dispersal patterns were similar at both scales, with non-contiguous dispersal responsible for colonization of new populations. High levels of differentiation were observed at all scales, though newly-colonized populations were more differentiated than older populations. Corvallis populations were responsible for colonization of a majority of populations throughout Oregon, while individuals from Eugene were only occasionally found in new populations. Admixture occurs between Corvallis and Eugene populations, decreasing differentiation, and potentially creating novel phenotypes and increasing evolutionary potential of populations. Selfing rates were high, but two populations in the areas of original introduction had lower rates of selfing, suggesting that selfing rates may decrease as population density and diversity increases with age. The influences of founder effects and bottlenecks on phenotypic evolution during range expansion require further investigation, as inbreeding, lag times, and selection may influence evolutionary trajectories of populations.

Understanding of grass genome structure and evolution has been significantly advanced through comparative genomics. The genomes of most cool-season forage and turf grasses, belonging to the Pooideae subfamily of the grasses, remain understudied. Creeping bentgrass (Agrostis stolonifera) is one of the most important cool-season turfgrasses due to its low mowing tolerance and aggressive growth habit. An RFLP genetic map of creeping bentgrass using 229 RFLP markers derived from cereal and creeping bentgrass EST-RFLP probes was constructed for a comparative genome analysis. This genetic map was compared with those of perennial ryegrass, oat, wheat, and rice. Large-scale chromosomal rearrangements between the map of creeping bentgrass and the respective maps of the Triticeae, oat, and rice were observed. However, no evidence of chromosomal rearrangements between the maps of creeping bentgrass and perennial ryegrass was detected, suggesting that these recently domesticated species might be closely related than expected. Further comparative genome analysis of creeping bentgrass was performed with the genome sequences of Brachypodium distachyon using sequences of the above-mentioned RFLP mapped markers and 8,470 publicly available A. stolonifera EST (AgEST) sequences. A total of 24 syntenic blocks were identified between the Agrostis linkage groups and the B. distachyon chromosomes. Orthologous loci of AgESTs (678) were identified in the B. distachyon genome, and these loci can be utilized in further comparative mapping of Pooideae species. Insights from comparative genomics with B. distachyon will be useful for genetic improvement of Agrostis spp. and provide a better understanding of the evolution of the Pooideae species.

La fusariose des épis est l’une des principales maladies des céréales, majoritairement causée par le champignon pathogène et toxinogène, Fusarium graminearum (Fg). Lors son développement in planta, le champignon produit des mycotoxines dommageables pour la santé humaine et animale, dont le déoxynivalénol (DON). De nombreux loci à effet quantitatif sur la résistance à Fg ont été identifiés chez le blé tendre. Certains d’entre eux ont été corrélés à la capacité à détoxifier le DON, en particulier par glucosylation sous l’action d’UDP-glucosyltransférases (UGT). Une UGT d’orge impliquée dans la conjugaison du DON a été identifiée en système hétérologue. Brachypodium distachyon (Bd) a récemment émergé comme modèle d’étude pour les céréales. Ce travail à l’aide d’approches transcriptomique et métabolomique a mis en évidence que lors de l’interaction avec Fg, Bd met en place des réponses macroscopiques, moléculaires et métaboliques similaires à celles connues chez le blé et l’orge. La recherche d’UGTs candidates capables de conjuguer le DON en DON-3-O-glucoside (D3G) chez Bd a permis l’identification d’un candidat. L’analyse fonctionnelle du gène correspondant a été conduite par des approches de mutagenèse et de surexpression. Ceci a montré une sensibilité accrue des lignées mutantes à la toxine et à l’agent pathogène. A l’inverse les lignées surexpresseurs ont montré une tolérance et résistance quantitative à la toxine et l’agent pathogène. Ces résultats ont été corrélés par la détection in planta de DON et D3G, dans des proportions variables selon les lignées. Ces résultats démontrent le rôle majeur que joue la glucosylation du DON dans l’établissement de la résistance observée chez Bd en réponse à Fg. / Fusarium head blight is a major cereal disease, mostly caused by the pathogenic and toxin-producing fungus, Fusarium graminearum (Fg). During its development in planta, the fungus produces mycotoxins harmful to human and animal health, including deoxynivalenol (DON). Many quantitative trait loci exhibiting an effect on resistance to Fg have been identified in wheat. Some of them were correlated with the ability to detoxify DON, particularly by glucosylation by UDP-glycosyltransferases (UGT). A barley UGT involved in the conjugation of DON was identified in a heterologous system. Brachypodium distachyon (Bd) has recently emerged as a model species for cereals. Using transcriptomic and metabolomic approaches, we show that when interacting with Fg, Bd implements macroscopic, molecular and metabolic responses similar to those known in wheat and barley. The search for UGT candidates able to conjugate DON into DON-3-O-glucoside (D3G) in Bd resulted in the identification of the Bradi5g03300 gene. Functional analyses of this gene showed increased sensitivity of the mutant lines to the toxin and to the pathogen. Conversely the overexpressor lines showed a tolerance to the toxin and quantitative resistance to Fg. These results were correlated with the detection of differential amounts of DON and D3G in the different lines. These results demonstrate the important role of DON glucosylation in the resistance establishment of Bd observed in response to Fg.

Transcriptomics and gene expression profiling enables the elucidation of the genetic response of an organism to various environmental cues. Transcriptomics enables the deciphering of differences between two closely related organisms to the same environment and in contrast, enables the elucidation of genetic responses of the same organism to different environmental cues. Two major methods are utilized for the study of transcriptomes, high-throughput sequencing and microarray analysis. High-throughput sequencing technologies such as the Illumina platform are relatively new and protocols must be developed for the analyses of transcriptomes (RNA-sequencing). A RNA-seq protocol was developed and refined for the Illumina sequencing platform. This protocol was then utilized for the de novo sequencing of the steelhead salmon transcriptome. Hatchery steelhead exhibit a reduced fitness compared to wild steelhead that has been shown to be genetically based. Consequently, the steelhead transcriptome was assembled, annotated, and used to identify gene expression differences between hatchery and wild fish. We uncovered many differentially expressed genes involved in metabolic processes and growth and development. This work has created a better understanding of the genetic differences between hatchery and wild steelhead salmon. Brachypodium distachyon is a monocot grass important as a model for cereal crops and potential biofuels feedstocks. To better understand the genetic response of this plant to different environmental cues, a comprehensive assessment of the transcriptomic response was conducted under a variety of conditions including diurnal/circadian light/dark/temperature environments and different abiotic stress conditions. Using a whole-genome tiling DNA microarray, we identified that the majority of transcripts in Brachypodium exhibit a daily rhythm in their abundance that is conserved between rice and Brachypodium. We also identified numerous cis-regulatory elements dictating these rhythmic expression patterns. We also identified the genetic response to abiotic stresses such as salinity, drought, cold, heat, and high light. We uncovered a core set of genes which responds to all stresses, indicating a core stress response. A large number of transcription factors were uncovered as potential nodes for regulating the abiotic stress response in Brachypodium. Moreover, promoter elements that drive specific responses to discrete abiotic stresses were uncovered. Altogether, the transcriptome analyses in this work furthers our understandings of how particular organisms respond to environmental cues and better elucidates the relationship between genes and the environment. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Oct. 5, 2011 - April 5, 2012.

Les lignines sont des hétéropolymères phénoliques de la paroi cellulaire, principalement à base de p-coumaryl, coniférylique et sinapylique alcools. Ces monolignols sont synthétisées dans le cytoplasme de la voie des phénylpropanoïdes, peut ensuite transportées vers les parois des cellules où ils sont polymérisés par oxydation en p-hydroxyphényl (H), guaiacyle (G) et syringyle (S) des unités de lignine. Cette étape de polymérisation par oxydation est conduite par les peroxydases dépendantes H₂O₂ et / ou laccases dépendantes O₂. Dans cette étude, nous avons signalé pour la première fois que les laccases sont également impliqués dans la lignification du les herbes. Un gène de laccase spécifique (BdLAC5) a été identifié parmi les 29 gènes de laccase non redondants dans Brachypodium génome, qui est responsable de la lignification dans les tiges de Brachypodium distachyon, une plante modèle pour les graminées. BdLAC5 gène a été retrouvé fortement exprimé dans les organes lignifiées (internodal, nœud et le pédoncule) et mal exprimé dans les organes avec faible niveau de lignine (jeunes feuilles et épillet), ni dans les tissus non-lignifiée (endosperme). Deux autres laccases BdLAC6 et BdLAC8 sont également trouvés coexprimés avec BdLAC5 et curieusement ils appartiennent à la même clade phylogénétique. BdLAC6 et BdLAC8 sont orthologues de Arabidopsis LAC4 et LAC2 respectivement. Dans les expériences d'hybridation in situ ont démontré le signal le plus intense dans les fibres interfasciculaires de l'entre-nœud a été détectée avec des sondes BdLAC5. En outre, des essais ont révélé que les protéines immunomarquages BdLAC5 pourraient être sécrétés dans la matrice de la paroi cellulaire, car nous avons détecté des particules fluorescentes dans ou à proximité de la paroi cellulaire. Le double mutant laccase touchée BdLAC5 et BdLAC8 a clairement montré que la lignification dans les fibres interfasciculaires impliqué différents gènes / protéines que la lignification dans les cellules métaxylème de Brachypodium. Métaxylème cellules ont été que faiblement affectés dans le double mutant lorsque les fibres interfasciculaires montré diminution dramatique de Wiesner coloration. Les différents mécanismes de lignification entre xylème et fibres est discutée. L'interaction physique et la synergie entre réglementation spécifique R2R3-MYB, bHLH et WDR protéines est bien étudiée dans la biosynthèse des flavonoïdes, racine des cheveux, trichomes et le développement en mucilage de graines de différentes espèces de plantes. Dans cette étude, nous avons essayé de comprendre les rôles de MYB-bHLH-WDR pour la régulation de la biosynthèse de la lignine. / Lignins are cell wall phenolic heteropolymers, mainly made from p-coumaryl, coniferyl, and sinapyl alcohols. These monolignols are synthesized in the cytoplasm from the phenylpropanoid pathway, then may transported to the cell walls where they are oxidatively-polymerized into p_hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units. This oxidative polymerization step is driven by H₂O₂-dependent peroxidases and/or O₂-dependent laccases. In this study we reported for the first time that laccases are also involved in lignification in grasses. A specific laccase gene (BdLAC5) was identified among 29 non-redundant laccase genes in Brachypodium genome, which is responsible for the lignification in stems of Brachypodium distachyon, a model plant for grasses. BdLAC5 gene was found highly expressed in lignified organs (internode, node and peduncle) and poorly expressed in organs with low lignin level (young leaf and spikelet) nor in non-lignified tissue (endosperm). Two other laccases BdLAC6 and BdLAC8 are also found coexpressed with BdLAC5 and interestingly enough they belong to the same phylogenetic clade. BdLAC6 and BdLAC8 are close orthologues of Arabidopsis LAC4 and LAC2 respectively. In situ hybridization experiments demonstrated the most intense signal in the interfascicular fibers of the internode was detected with BdLAC5 probes and then for BdLAC8 and BdLAC6 probes. Furthermore, immunolabelling assays revealed that BdLAC5 proteins might be secreted into the cell wall matrix because we detected some fluorescent particles close to or in the cell wall. The double laccase mutant affected in BdLAC5 and BdLAC8 (5ho8ho) clearly showed that the lignification in interfascicular fibers involved different genes/proteins than the lignification in metaxylem cells of Brachypodium. Metaxylem cells were only poorly affected in the double mutant when interfascicular fibers showed dramatic decrease of Wiesner staining. The different mechanisms of lignification between xylem and fibers is discussed. The physical interaction and regulatory synergy between specific R2R3-MYB, bHLH and WD repeat protein is well studied in the biosynthesis of flavonoids, root hair, trichome and seed mucilage development in different plant species. In this study, we were trying to figure out the roles of MYB- bHLH-WDR for the regulation of lignin biosynthesis.