Trichome formation in Arabidopsis lyrata is a naturally occurring trait with phenotypic polymorphisms within wild populations. In Swedish accessions of A. lyrata, three genetic polymorphisms situated in the coding region of GL1, an important transcription factor in trichome production, have been identified, and these are candidates for being the cause of a glabrous phenotype. In this study a complementation test has been performed to clarify which mutation/mutations that are detrimental for trichome formation. A set of constructs has been transformed into A. thaliana, a close relative to A. lyrata, and subsequent generations of plants were examined for phenotype, genotype and gene expression. A SNP (Single Nucleotide Polymorphism) in the R3 MYB domain of GL1, resulting in a change of an alanine to aspartic acid, was identified as the critical polymorphism. The other two mutations, two indels, were harmless to protein function. The inserted constructs were under control of the native GL1 promoter. Plants that, because of the SNP, lacked trichome production, became totally glabrous.

Orientador: Michel Georges Albert Vincentz / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T03:47:47Z (GMT). No. of bitstreams: 1 Silveira_AmandaBortolini_D.pdf: 6025388 bytes, checksum: 97930bc131f2a0eac6926dae1eeb9319 (MD5) Previous issue date: 2012 / Resumo: Modificações epigenéticas do DNA ou da cromatina atuam principalmente no controle da atividade de elementos de transposição, podendo também silenciar genes, geralmente quando estes estão associados a elementos de transposição ou sequências repetidas. Em plantas, alguns alelos epigenéticos afetando caracteres como morfologia floral, florescimento, estatura ou amadurecimento do fruto foram descritos, revelando o potencial deste tipo de regulação para gerar variabilidade fenotípica herdável não necessariamente vinculada a alterações da sequência de DNA. No entanto, o impacto de mecanismos epigenéticos em processos de evolução adaptativa é ainda bastante desconhecido, em parte, pela falta de informação sobre variação epigenética em populações naturais. Identificamos Qua-Quine Starch (QQS) de Arabidopsis thaliana como um gene sob um controle epigenético flexível e, portanto, particularmente propenso a variações epialélicas frequentes na natureza. QQS é um gene recente, que provavelmente originou-se de novo em Arabidopsis thaliana em uma região rica em elementos de transposição. Mostramos que QQS apresenta-se diferencialmente expresso entre acessos naturais assim como entre indivíduos diretamente coletados na natureza e que estas diferenças de expressão estão negativamente correlacionadas com o nível de metilação de sequências repetidas localizadas em sua região promotora e 5' UTR, não estando relacionadas a variação genética em cis ou trans. Mostramos ainda que variação epialélica em QQS é independente do nível de metilação de transposons vizinhos e que pode ser estavelmente herdada entre gerações. Considerando o impacto potencial de padrões de expressão contrastantes de QQS no metabolismo de amido, um importante componente para produção de biomassa e crescimento, sugerimos que variação epialélica em QQS possa ter implicações adaptativas. Nossos dados também apontam pela primeira vez uma ligação potencial entre mecanismos epigenéticos e o processo de evolução de genes novos. Propomos que genes novos, especialmente os de origem de novo, poderiam ser mais propensos a variar epigeneticamente, o que permite um ajuste fino de seu padrão de expressão até que o estado mais vantajoso seja fixado geneticamente / Abstract: Epigenetic modifications of DNA or chromatin control of the activity of transposable elements and can also silence genes which are associated to transposons or repetitive sequences. In plants, epigenetic alleles affecting characters such as floral morphology, flowering, stature or fruit ripening have been described, highlighting the potential of this type of regulation in generating heritable phenotypic diversity, not necessarily linked to DNA sequence alterations. However, the impact of epigenetic mechanisms in adaptative evolution is still largely unknown, in part, due to the lack of information about epiallelic variation in natural populations. We have identified Qua-Quine Starch (QQS) of Arabidopsis thaliana as a gene under flexible epigenetic control and thus particularly prone to epiallelic variation in nature. QQS is a recent gene that likely originated de novo in Arabidopsis thaliana in a transposon-rich region. We show that QQS is differentially expressed among natural accessions as well as among individuals directly sampled from the wild and that these expression differences are negatively correlated with the DNA methylation level of repeat sequences located on QQS promoter and 5'UTR region and are not correlated with cis or trans genetic variation. We also show that epiallelic variation at QQS is independent of the methylation status of nearby transposable elements and can be stably inherited across generations. Considering the potential impact of contrasting QQS expression patterns on starch accumulation, an important component of biomass production and growth, we suggest that epiallelic variation at QQS may have adaptative implications. Our data also points for the first time to a potential link between epigenetic mechanisms and the evolution of novel genes. We suggest that novel genes, more specifically those created de novo, could be endowed with an increased potential for epigenetic variation and thus for adjusting their expression pattern until the most adaptive state becomes genetically fixed / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Les polysaccharides sont des composants majeurs des parois cellulaires ayant une structure dynamique et jouant un rôle essentiel dans la croissance des plantes. Les cellules épidermiques du tégument des graines d’Arabidopsis libèrent un halo de mucilage polysaccharidique lors de leur imbibition. Le mucilage séminal s'est avéré être un excellent système modèle pour l’étude de la production, des propriétés des polysaccharides et de leurs interactions. Le premier objectif de ma thèse était de valoriser la variabilité naturelle existant entre accessions d’Arabidopsis pour identifier de nouveaux gènes contrôlant la production de mucilage. Une analyse de génétique d’association a permis l’identification d’une amine oxidase et d’une glycosyltransferase putatives dont j’ai confirmé l’implication dans la biosynthèse des pectines du mucilage.J’ai également associé une famille de protéines aux interactions entre polysaccharides. De part ses propriétés d’hydrogel, le mucilage joue un rôle adaptatif et influence la physiologie de la graine. Il permet la rétention d’eau autour de celle-ci et pourrait ainsi influencer sa longévité. Le deuxième objectif de ma thèse était d’utiliser des mutants impactés dans la production de mucilage pour déterminer si ce dernier influence la longévité des graines après un traitement d’hydratation contrôlée appelé ‘priming’. Les graines ne libérant pas de halo de mucilage à l’imbibition ont une meilleure longévité en lien avec une diminution réduite d’acide salicylique. Mes résultats participent à une compréhension intégrée de la production de mucilage à plusieurs niveaux : écologiques, génétiques et physicochimiques. / Polysaccharides are the major component of cell walls that are dynamic structures playing a fundamental role in plant growth. On imbibition, the epidermal cells of the Arabidopsis seed coat release a mucilage hydrogel formed of polysaccharides. This has proved to be an excellent model system for the study of cell wall polysaccharide production, properties and interactions. The first objective of my thesis was to exploit natural variation between Arabidopsis accessions to identify genes controlling mucilage polysaccharide production. A genome wide association study identified genes encoding proteins with putative functions as either an amine oxidase or glycosyltransferase and these were confirmed to contribute to the synthesis of mucilage pectin. I also found that a family of small proteins, whose function is undetermined, are likely to modulate the interaction of mucilage polymers. Mucilage is also an adaptive trait that may influence various aspects of seed physiology. Recent results indicate that this hydrogel plays a role in the retention of water around the seed and could influence their lifespan. A second objective of my thesis was to use mutants showing altered mucilage production to determine its contribution to seed lifespan after a controlled hydration treatment called ‘priming’. Seeds that do not release mucilage on imbibition retained longevity better after priming. I highlighted that the steady state levels of salicylic acid in primed seeds were influenced by mucilage and correlated negatively with their longevity. My results contribute to our genetic, physicochemical and ecophysiological understanding of mucilage production by seeds.

No description available.

Organisms are often adapted to their local environment, but the role of early life stages in adaptive differentiation among populations remains poorly known. The aim of my thesis was to investigate the contribution of early life stages to the magnitude and genetic basis of local adaptation, and to identify the underlying adaptive traits. For this, I used two natural populations of the annual plant Arabidopsis thaliana from Italy and Sweden, and a Recombinant Inbred Line (RIL) population derived from a cross between these populations. By combining greenhouse and field experiments, Quantitative Trait Loci (QTL) mapping, and path analysis, I examined (1) the genetic basis of seed dormancy, (2) the contribution of differential seedling establishment to local adaptation, (3) among-year variation in selection during seedling establishment, (4) direct and indirect effects of seed dormancy and timing of germination on fitness, and (5) the adaptive value of the seed bank. I found that both the level and the genetic basis of seed dormancy were affected by the maternal environment. One major-effect QTL was identified in all maternal environments, which overlaps with the dormancy gene DELAY OF GERMINATION 1 (DOG1). Selection through seedling establishment success contributed strongly to local adaptation and genetic tradeoffs, and varied among years. Variation in seedling establishment and overall fitness among RILs could be explained by genetically based differences in seed dormancy and timing of germination. Seed dormancy affected fitness throughout the life cycle, by affecting the proportion of germinated seeds, and indirectly via effects on timing of germination, plant size and flowering time. My results suggest that a considerable portion of A. thaliana seeds enter the seed bank. I found genetic differences in dormancy cycling behaviour between the two populations, which could contribute to local adaptation. The value of a seed bank should be higher at the Swedish study site than at the Italian study site due to lower rate of seed mortality in the soil. Overall, the results of this thesis demonstrate that early life stages contribute strongly to both the magnitude and the genetics of local adaptation.

<p>Sorghum [<i>Sorghum bicolor</i> (L.) Moench] is a staple food for millions of people in Africa and South Asia. It is mainly consumed for its starch. The starch composition and structure in the seed endosperm determines cooking properties, processing quality, and starch digestibility. </p> <p>An assay to measure the alkali spreading value (ASV) of sorghum is described. The assay was used to identify sorghum EMS mutants with variation in starch composition. The ASV mutants (ASV+) exhibited a range of starch thermal properties with starch gelatinization temperatures (GT) being lower or higher than samples from Tx623 or Sepon82. The ASV+ phenotypes were found to be correlated with starch related traits such as enthalpy (r = −0.53) and range of starch GT (T_c-T_o) (r = 0.60). </p> <p>Genes controling the ASV phenotype of sorghum and their impact on starch quality traits are described. Whole genome re-sequencing of sorghum EMS mutants exhibiting an ASV+ phenotype was used to identify single nucleotide polymorphisms (SNPs) in candidate genes <i>Sobic.004G163700</i> and <i>Sobic.010G093400</i>. The two genes were identified as a <i>SbeIIb</i>, a putative sorghum homolog of <i>amylose extender,</i> and as a <i>SSIIa</i>, respectively. Linkage analysis showed that the mutations in <i>Sobic.010G093400</i> and <i>Sobic.004G163700</i> co-segregated with the ASV phenotype. The <i>ssIIa</i>-mutants exhibited normal amylose values, lower starch GT and lower final viscosity than the wild type. The <i>sbeIIb</i>-mutants exhibited higher amylose content, higher starch GT and lower peak and final viscosity with poor gel consistency compared to the wild type and <i>ssIIa</i>-mutants. An allele dosage test indicated that the <i>sbeIIb</i>-mutants had an allele dosage dependent effect on amylose content. Double mutants of <i>sbeIIb</i> and <i>ssIIa</i> showed that amylose content, starch thermal properties and paste viscosity profiles resemble the <i>sbeIIb</i> parent. </p> <p>A study of ASV phenotypes in a panel of more than 750 sorghum conversion lines revealed genetic variation for the ASV phenotype. A few SC-lines exhibiting stable expression of the ASV+ phenotype over two growing seasons. Most of these lines were described as belonging to the working group Nandyal, durra types from India described as producing ‘glutinous grains’. Whole genome resequencing discovered common SNPs in genes associated with starch biosynthesis. A genome wide association study (GWAS) identified a significant SNP that could be associated with the starch biosynthesis gene <i>Sobic.010G273800</i>, and with candidate genes <i>Sobic.010G274800</i> and <i>Sobic.010G275001</i> both annotated as glucosyltransferases. Grain samples from SC489, SC491, SC587 and SC589 exhibited a consistent ASV+ phenotype with lower or similar starch GT, similar amylose content, and similar high viscosity and gel consistency compared to controls.</p> <p> </p>

Le réchauffement climatique constitue une problématique d'actualité très préoccupante en raison de ses effets potentiels sur la biodiversité et le secteur agricole. Mieux comprendre l'adaptation des plantes face à ce phénomène récent représente donc un intérêt majeur pour la science et la société. L'étude de populations naturelles provenant d'un gradient d'altitude permet de corréler l'impact d'un ensemble de conditions climatiques (température, humidité, radiation, etc.) à des traits phénotypiques. Ces différentes populations sont dites adaptées à leurs conditions climatiques in natura. En cultivant ces plantes dans des conditions standardisées de laboratoire (intensité lumineuse, substrat, température, arrosage, etc...), la variabilité phénotypique observée, est alors due essentiellement à la variabilité génétique intrinsèque à chaque plante, donc à son génotype. La mise en culture de ces mêmes plantes en changeant une seule variable, par exemple la température, permet de mettre en évidence un phénotype caractéristique. Ce phénotype observé peut être une réponse d'acclimatation d'un génome adapté. Le projet WallOmics vise à caractériser l'adaptation des plantes à l'altitude par l'étude de populations naturelles d'Arabidopsis thaliana provenant des Pyrénées. Les acteurs moléculaires de l'adaptation des plantes au climat sont encore mal connus mais il apparaît que la paroi des cellules végétales pourrait avoir un rôle important dans ce processus. En effet, celle-ci représente le squelette des plantes et leur confère une rigidité tout en représentant une barrière externe sensible et dynamique face aux changements environnementaux. Sa structure et sa composition peuvent être modifiées à tout moment. Il est d'ailleurs possible de dire que cette paroi végétale donne la forme générale de la plante (taille, forme, densité, etc...), son phénotype observable. Ce projet se consacrera principalement à l'étude des parois des cellules végétales. Les nouvelles technologies ont permis l'émergence des données dites "omiques", c'est-à-dire de vastes ensembles de données provenant de niveaux biologiques multiples, comme des données écologiques, de phénotypages, biochimiques, protéomiques, transcriptomiques et génomiques. L'étude et la mise en relation de ces données ont favorisé le développement d'approches globales qui visent à établir une réponse à plusieurs échelles. C'est justement par ce type d'approche non mécanistique que le projet WallOmics a contribué à établir les bases moléculaires des modifications des parois face aux changements climatiques. / Global warming is a current issue of great concern because of its potential effects on biodiversity and the agricultural sector. Better understanding the adaptation of plants to this recent phenomenon is therefore a major interest for science and society. The study of natural populations from an altitude gradient allows correlating a set of climatic conditions (temperature, humidity, radiation, etc...) with phenotypic traits. These different populations are considered as adapted to their climatic conditions in natura. By cultivating these plants under standardized laboratory conditions (light intensity, substrate, temperature, watering, etc.), the observed phenotypic variability, is essentially due to the genetic variability intrinsic to each genotype. The growth of these same plants by changing a single variable, for example temperature, makes possible to highlight a characteristic phenotype. This phenotype may be an acclimation response of a relevant genome. The WallOmics project aims at characterizing the adaptation of plants to altitude by studying natural populations of Arabidopsis thaliana from the Pyrenees. The molecular actors of the adaptation of plants are still poorly described, but it appears that the plant cell wall could play an important role in this process. Indeed, it represents the skeleton of plants and gives them rigidity while representing a dynamic and sensitive external barrier to environmental changes. Its structure and composition can be modified at any time. It is also possible to say that the plant cell wall gives the general shape of the plant (size, shape, density, etc.), that is its observable phenotype. This project will focus mainly on the study of the plant cell wall. New technologies have enabled the emergence of the so-called "omics" data, large sets of data at multiple biological levels, such as ecological, phenotypic, metabolomic, proteomic, transcriptomic and genomic data. The study and the links between these data have favoured the development of integrative approaches aimed at establishing a response at several scales. It is precisely by this type of non- mechanistic approach that the WallOmics project has contributed to establish the molecular players of plant cell wall modifications in the global warming context.

<p dir="ltr">Communication between the male gametophyte (the tip-growing pollen tube) and the female gametophyte (the synergid cells) is crucial for sexual reproduction in flowering plants. The reception of the pollen tube (PT) depends on its recognition and sensitivity to the signals from the synergid cells for its rupture, sperm release, and double fertilization. Mutations in genes regulating communication between the synergid cells and pollen tube lead to PT overgrowth. Despite significant advances in understanding the molecular mechanism of pollen tube reception in Arabidopsis, there remains a need for more comprehensive information on the impact of natural variations on physiological traits related to pollen tube and synergid signals in pollen tube reception. This research investigates the effects of natural variation on pollen tube sensitivity to synergid signals mediated by NORTIA. In <i>nortia</i> homozygous mutants, PT-synergid communication is disrupted due to lower levels of calcium signals in the synergid cells, resulting in PT overgrowth. Using the Aniline blue staining procedure, this study identified twelve ecotypes of Arabidopsis thaliana out of the twenty-four ecotypes that could suppress the PT overgrowth phenotype of <i>nta-1</i>. I observed that the suppressor ecotypes exhibit characteristics like the Columbia (Col-0) ecotype, while non-suppressor ecotypes resemble the Ws-2 ecotype. Comparing the impact of the Columbia (Col-0) and Wassilewskija (Ws-2) ecotypes on PT overgrowth in the <i>nta-2</i> mutant revealed that Columbia (Col-0) effectively suppressed PT overgrowth compared to Wassilewskija (Ws-2). We investigated the sensitivity of PT integrity mutants to synergid signals. Our results showed that compromised PT integrity mutant genes (<i>mlo1mlo15</i>) partially suppressed PT overgrowth in <i>nta-1</i>. I propose that suppressor ecotypes and mutants may exhibit a heightened sensitivity to synergid signals, that help them to regulate their response to synergid signals finely.</p>

Genotypes adapted to contrasting environments are expected to behave differently when placed in common controlled conditions, if their sensitivity to environmental cues or intrinsic growth behaviour are set to different thresholds, or are limited at distinct levels. This allows natural variation to be exploited as an unlimited source of new alleles or genes for the study of the genetic basis of quantitative trait variation. My doctoral work focuses on analysing natural variation for shoot growth and response to the environment in A. thaliana. Natural variation analyses aim at understanding how molecular genetic or epigenetic diversity controls phenotypic variation at different scales and times of plant development and under different environmental conditions, and how selection or demographic processes influence the frequency of those molecular variants in populations for them to get adapted to their local environment. As such, the analysis of A. thaliana natural variation can be addressed using a variety of approaches, from genetics and molecular methods to ecology and evolutionary questions. During my PhD, I got the chance to tackle several of those aspects through my contributions to three independent projects which have in common to exploit A. thaliana natural variation. The first one is the analysis of the pattern of polymorphism from a set of 102 A. thaliana accessions at the MOT1 gene coding for a molybdate transporter (an essential micronutrient) and responsible for contrasted growth and fitness among accessions in response to Mo availability in the soil. I showed at different geographical scales that MOT1 pattern of polymorphisms is not consistent with neutral evolution and shows signs of diversifying selection. This work helped reinforce the hypothesis that in some populations, mutations in MOT1 have been selected to face soils rich in Mo and potentially deleterious despite their negative effect on Mo-limiting soils. The second project consists in the characterisation and functional analysis of two putative receptor-like kinases (RLKs) identified from their effect on shoot growth specifically under mannitol-supplemented media and not in response to other osmotic constraints. The function of such RLKs in A. thaliana, which is not known to synthesize mannitol was intriguing at first but, through different experiments, we built the hypothesis that those RLKs could be activated by the mannitol produced by some pathogens such as fungi and participate to plant defensive response. The third project, in collaboration with Michel Vincentz's team from CBMEG (Brasil) and Vincent Colot (IBENS, Paris), consists in the analysis of the occurrence of natural epigenetic variants of the QQS gene in different populations from Central Asia and their possible phenotypic and adaptive consequences. Overall, these analyses of the genetic and epigenetic molecular variation leading to the biomass phenotype(s) in interaction with the environment provide clues as to how and where in the pathways adaptation is shaping natural variation.

Les microsporidies sont des pathogènes intracellulaires obligatoires apparentés aux champignons. Elles infectent de nombreux animaux, dont le nématode Caenorhabditis elegans. La première microsporidie isolée d’une souche de C. elegans sauvage a été nommée Nematocida parisii. L’interaction entre N. parisii et C. elegans est devenue un puisant modèle pour l'étude des interactions hôte-pathogène. Cependant, ce modèle a été récemment découvert et de nombreux détails sur son écologie et sa biologie restaient inconnus. Notamment, nous ignorions l’incidence et la diversité des infections microsporidiennes chez C. elegans et autres nématodes dans la nature.A partir d’une collection de nématodes, de la famille des Rhabditidae, échantillonnés dans le monde entier, j’ai recensé un panel de 47 nématodes présentant des symptômes d’infection par des microsporidies. J’ai caractérisé moléculairement la diversité de ce parasite infectant ces nématodes et déterminé que N. parisii est la microsporidie la plus souvent responsable des infections chez C. elegans dans la nature. J’ai également décrit et nommé six nouvelles espèces de Nematocida. Au cours de mes travaux, j’ai aussi défini deux nouveaux genres de microsporidies génétiquement distincts de Nematocida, appelés Enteropsectra et Pancytospora. Mes travaux ont de plus détaillé la diversité qui existe chez les microsporidies parasites de nématodes. Ces microsporidies présentent des différences en terme de taille et forme de leurs spores, de leur tropismes tissulaire et intracellulaire chez l’hôte, de leur voie de sortie des cellules hôtes mais aussi de spectre d’hôtes. Mes résultats ont démontré que, dans la nature, les infections de C. elegans et autres nématodes par les microsporidies sont répandues et diverses.De plus, j’ai estimé la variation naturelle pour la sensibilité de C. elegans à l'infection par N. ausubeli. J’ai notamment comparé 10 souches naturelles de C. elegans en utilisant des tests de consommation alimentaire. Deux souches de C. elegans, JU1249 et JU2825, présentaient des niveaux contrastés de sensibilité, ce que j’ai interprété comme étant une différence de niveau de tolérance aux infections. Ces deux souches se sont révélées être de bons candidats pour une future caractérisation des loci génétiques associés à la variation de sensibilité de C. elegans aux infections microsporidiennes. Enfin, j’ai observé un effet surprenant de l'infection de C. elegans par les microsporidies. En effet, la présence du pathogène est capable de supprimer le déclin progressif de la fécondité à haute température chez certaines lignées de C. elegans. / Microsporidia are fungi-related intracellular pathogens that infect a great variety of animals, including the nematode Caenorhabditis elegans. The first microsporidia isolated from wild C. elegans was named Nematocida parisii in 2008. C. elegans and N. parisii have been used as a powerful model for the study of host-pathogen interactions. However, it was unclear how widespread and diverse microsporidia infections are in C. elegans or other related nematodes in the wild.By sampling rhabditid nematodes worldwide, we established a collection of 47 nematodes that displayed putative microsporidia infections. We characterized molecularly these infections and determined that N. parisii (or N. ironsii) is the most common microsporidia infecting C. elegans in the wild. We further described and named six new Nematocida species. In addition, we defined two new genera of nematode-infecting microsporidia, named Enteropsectra and Pancytospora, which are genetically distinct from Nematocida. Further investigations showed that these microsporidia are diverse in terms of spore size and shape, host tissue tropism, host cell intracellular localization, cellular exit route, host specificity pattern, etc. Overall, these findings illustrate the widespread and diverse microsporidia infections in C. elegans and related nematodes in the wild.We further assayed the natural variation of C. elegans in sensitivity to N. ausubeli infection, by comparing 10 C. elegans strains using food consumption tests. Two C. elegans strains, JU1249 and JU2825, displayed the largest sensitivity differences, which were suggested to be a result of the different tolerance between the two strains. These two strains are proven to be good candidates for future studies on the genetic loci associated with C. elegans sensitivity variation to microsporidian infections. Furthermore, I observed an exciting effect of host-pathogen interaction. Microsporidia infection is able to suppress the progressive decline in fertility in some C. elegans with the mortal germline phenotype (Mrt).