Chez Arabidopsis thaliana, Les gènes d'ARN 5S sont regroupés en blocs situés dans l'hétérochromatine péricentromérique des chromosomes 3,4 et 5. La transcription des gènes d'ARN 5S est régulée par des facteurs épigénétiques altérant notamment leur structure chromatinienne. Une étude a été menée durant les premières étapes du développement post-germinatif pour identifier les évènements et des facteurs conduisant à l'élaboration de telles structures. Nous avons pu observer une décompaction de l'ADNr 5S immédiatement suivie d'une recondensation. Ces phénomènes impliquent respectivement ROS 1 et l'ARN polymérase IV. L'étude des formes Pol IVa et Pol IVb nous indique que Pol IVb, en plus de son activité partenaire de Pol IVa, possède une action spécifique dédiée au locus d'ADNr 5S du chromosome 4. Cette nouvelle activité de Pol IVb, qui est indispensable au silencing et à la compaction de ce locus, semble indépendante de la voie classique de méthylation de l'ADN dépendante des ARN.

L'étude des éléments cis et trans intervenant dans le métabolisme des ARN SINE a pour but de mieux appréhender la biologie des éléments mobiles SINE. Nous avons d'une part déterminé la structure secondaire des ARN des SINE de plante SB1 et SB2. Ces deux ARN qui n'ont pas d'homologie de séquence adoptent des structures secondaires similaires. Suite à cette observation, une étude menée par F.J. Sun et al. a mis en évidence un schéma évolutif commun des ARN SINE dérivés d'ARNt. Dans le cadre de la recherche de facteurs trans, nous avons entrepris la caractérisation de la protéine La, un facteur de liaison à l'ARN. Nous avons identifié chez Arabidopsis deux protéines présentant toutes les caractéristiques de la protéine La : At32 et At79. Nous avons montré que seule At32 assure les fonctions nucléaires de la protéine La. At32 et At79 ont des profils d'expression différents et semblent lier des ARN distincts. Nous proposons donc qu'il existe deux homologues de la protéine La Chez Arabidopsis

This study investigated whether participation in the Partnership for Research and Education in Plants (PREP), a long-term authentic plant research project, in conjunction with explicit verses implicit instruction can change high school students' conceptions of the nature of science (NOS). The participants included a total of 134 students comprised of three groups from 10 total classes over the course of two academic years. Participants in four classes (two each year) participated in PREP and received explicit instruction on NOS. Participants in four other classes (two each year) participated in PREP and received implicit only instruction on NOS. Additionally, two classes (one each year) of high-achieving freshmen participated in PREP and received explicit instruction on NOS. This third group was used as a comparative group to the other two groups, due to their high achievement in middle school math and science. The treatment for all three groups spanned 8 weeks and included participation in an authentic plant research project. An open-ended questionnaire (modified Views of Nature of Science - VNOS), in conjunction with semi-structured interviews, was used to assess students' conceptions before and after the intervention. Results showed that all three groups improved their conceptions of NOS equally. The high-achieving group began with significantly higher-scoring views prior to the completion of the intervention, and improved to the same degree as the other two groups. A comparison of the explicit group to the implicit only group showed that there was no significant difference in their improvement, as both groups improved equally. Implications for the teaching and learning of NOS are discussed.

Ribosomes, large enzymatic complexes containing an RNA catalytic core, drive protein synthesis in all living organisms. 80S cytoplasmic eukaryotic ribosomes are comprised of four rRNAs and approximately 80 ribosomal proteins (r-proteins). R-proteins are encoded by gene families with large families (average of twelve members) predominating in mammals and smaller families (two to seven members) in plants. Increased ribosome heterogeneity is possible in plant ribosomes due to multiple transcriptionally active members in each family, whereas, in mammalian r-protein gene families, only one member is typically active. Multiple functional paralogs provide for greater plasticity in response to environmental/developmental cues, as well as, increasing the possibility of individual paralogs procuring or retaining extraribosomal functions. This research investigated the effects of r-protein mutations on plant growth and development. Through RNA interference (RNAi) mediated knockdown (KD) of type I (cytoplasmic: RPS15aA/D and F) and type II (non-cytosolic: RPS15aB and E) RPS15a family members I was able to confirm the delineation between the two types. Subcellular localization of the type I isoforms was nuclear/nucleolar while localization of type II isoforms was non-mitochondrial and probably cytosolic. Illumina sequencing of two r-protein mutant transcriptomes, pfl1 (rps18a) and pfl2 (rps13a), identified a novel set of up and down regulated genes, previously unknown or linked to r-protein mutants. The 20 genes identified were classified into four groups (1) plant defense, (2) transposable elements, (3) nitrogen metabolism and (4) genes with unknown function. Illumina miRNOME analysis revealed no changes in the miRNA profile of pfl1 and pfl2 plants. These data do not support the previously proposed theory that a disruption in ribosome biogenesis (by decreased r-protein synthesis) disrupts miRNA-mediated degradation of a range of auxin response genes. Finally, a novel double r-protein mutant, rps18a:HF/RPL18B, presented a late flowering/thickened bolt phenotype not seen in a rps13a:HF/RPL18B mutant, suggesting that RPS18A has an extraribosomal role in plant growth and development in Arabidopsis.

The plant phenylpropanoid pathway is initiated from deamination of phenylalanine to form cinnamic acid followed by hydroxylation and methylation of the aromatic ring to generate a variety of phenolic compounds including lignin monomers, flavonoid compounds and sinapate esters. The incorporation of phenylpropanoid metabolism served as a key step in the early land-colonization of plants from aqueous environment since phenolic compounds play important roles in plant development and abiotic/biotic stress responses. Lignin is a heteropolymer of hydroxycinnamyl alcohols that are derived from the major branch of plant phenylpropanoid pathway. The main function of lignin is to enhance the strength of plant cell wall and waterproof the vascular system for long-distance transportation of water and solutes. In addition, lignin is also involved in protecting plants against pathogen attack. My Ph.D. research is to investigate how lignin biosynthesis contributes to plant immunity. The results showed that the expression of major lignin biosynthetic genes was induced upon host fungal pathogen infection. Moreover, a mutant disrupted in the lignin gene F5H1 showed enhanced susceptibility when challenged with several fungal pathogens. F5H1 encodes a ferulic acid 5-hydroxylase that is uniquely present in angiosperm plants, leading to the biosynthesis of syringyl lignin monomer, which is not present in gymnosperm plants. Subsequent research demonstrated that f5h1 mutation impaired the penetration (pre-invasion) resistance but did not impact post-invasion resistance. Furthermore, the pathogen-induced expression of lignin genes was independent of well-characterized defensive signaling pathways, and regulated by a novel regulating mechanism. F5H1 contributes to pmr2-mediated resistance but acts independently of other molecular components of penetration resistance including PEN1, PEN2, and PEN3. In contrast to f5h1, a knockout mutant of flavonoid pathway gene chalcone isomerase (CHI/TT5) showed enhanced resistance to host anthracnose pathogen Colletotrichum higginsianum in a salicylic acid (SA)-dependent manner. Taken together, our results for the first time provide genetic evidence demonstrating that lignin biosynthetic gene F5H1 plays critical roles in plant penetration resistance and that an uncharted pathway in flavonoid metabolism confers an SA-dependent resistance pathway in Arabidopsis.

The link between the genotype and the phenotype of an organism is immensely complex. Despite this it can, to a great extent, be captured using models that assume that gene variants combine their effects in an additive manner. This thesis explores aspects of genetics that cannot be fully captured using such additive models. Using experimental data from three different model organisms, I study two phenomena that fall outside of the additive paradigm: genetic interactions and genetic variance heterogeneity. Using the model plant Arabidopsis thaliana, we show how important biological insights can be reached by exploring loci that display genetic variance heterogeneity. In the first study, this approach identified alleles in the gene CMT2 associated with the climate at sampling locations, suggesting a role in climate adaption. These alleles affected the genome wide methylation pattern, and a complete knock down of this gene increased the plants heat tolerance. In the second study, we demonstrate how the observed genetic variance heterogeneity was the result of the partial linkage of many functional alleles near the gene MOT1, all contributing to Molybdenum levels in the leaves. Further, we explore genetic interactions using data from dogs and budding yeast (Saccharomyces cerevisiae). In the dog population, two interacting loci were associated with fructosamine levels, a biomarker used to monitor blood glucose. One of the loci displayed the pattern of a selective sweep in some of the studied breeds, suggesting that the interaction is important for the phenotypic breed-differences. In a cross between two strains of yeast, with the advantage of large population size and nearly equal allele frequencies, we identified large epistatic networks. The networks were largely centered on a number of hub-loci and altogether involved hundreds of genetic interactions. Most network hubs had the ability to either suppress or uncover the phenotypic effects of other loci. Many multi-locus allele combinations resulted in phenotypes that deviated significantly from the expectations, had the loci acted in an additive manner. Critically, this thesis demonstrates that non-additive genetic mechanisms often need to be considered in order to fully understand the genetics of complex traits.

To identify novel and essential components of the plant membrane trafficking mechanisms, Arabidopsis membrane trafficking mutants from fluorescent protein-based forward genetic screens were characterized. First, four novel glutathione synthase (GSH2) mutant alleles featured swollen endoplasmic reticulum (ER)-derived bodies that accumulated a soluble secretory marker. Consistent with the role of GSH2 in glutathione biosynthesis, the loss-of-function mutant alleles exhibited gamma-glutamylcysteine (γ-EC) hyperaccumulation and glutathione deficiency. The aberrant ER morphology was ascribed to the γ-EC accumulation. Redox-sensitive fluorescent protein revealed that gsh2 seedlings maintained a reduced cytoplasm at steady state but were more sensitive to oxidative challenge. Second, Mut 21 was a conditional mutant that accumulated a secretory marker in the alkalized apoplast at restrictive temperature (31˚C). The mutant was identified as carrying a mutant allele of tuftelin-interacting protein 11 (TFIP11), which has been implicated in regulating redifferentiation and cell proliferation through a cytokinin signalling pathway. Hence, it was postulated that the changes in response to cytokinin affect auxin-mediated acidification of the apoplast. Third, Mut 43 was a conditional mutant that accumulated a soluble secretory marker in the ER and unidentified punctate structures at restrictive temperature, and exhibited perturbations in ER export of a soluble protein marker. Moreover, the mutant showed severe growth defects and abnormal radial root swelling in the apical elongation zone. A mutation identification method through deep-sequencing of the wild-type siblings in outcrossed heterozygous mutant families was developed and tested in Mut 43. At the time when this thesis was prepared, bioinformatic analysis has assigned Mut 43 to the bottom arm of chromosome two and predicted a 300kb mapping interval based on the observed bias in single nucleotide polymorphism ratios. This work demonstrates the feasibility of using forward genetics to study plant-specific aspects of membrane trafficking mechanisms and incorporates new technology to streamline the process of gene identification.

The Arabidopsis SON1 F-box protein was implicated in regulating a pathogen defense pathway, but its exact function in wild-type plants is unknown. As an F-box protein it was predicted that SON1 would assembles into a SON1-SCF ubiquitin ligase complex that recruits specific plant defense-related proteins for proteolysis. The yeast 2-hybrid assay was used to screen for potential substrates for a putative SON1-SCF ligase, leading to the identification of Arabidopsis LNK2 as a SON1-binding factor. Comprehensive protein-protein interaction analysis has shown that the binding of SON1 to LNK2 protein is specific, because closely related, full-length Arabidopsis F-box proteins do not interact with LNK2. However, when tested in isolation, some fragments derived from the paralog proteins did bind SON1, suggesting that higher order structure or inter-domain interference affects the ability of SON1 to recruit substrate. When analyzed for interaction domains, three regions of SON1 were identified that bind to LNK2 and a LNK2 binding region spans a conserved amino acid region. Phylogenetic analysis revealed that there is a paralogous gene called LNK1 in Arabidopsis, and both LNK1 and LNK2 are restricted to the plant kingdom. LNK2 and LNK1 are seen to be widely distributed in embryophyte seed and spore plants. Genetic analysis and complementation tests showed that LNK1 and LNK2 regulate flowering and photo-morphogenesis redundantly. Though lnk1 and lnk2 mutants look similar to wild-type plants, lnk1 lnk2 double mutant plants possess a long hypocotyls and flower late compared to wild-type plants. Because SON1 is implicated in plant defense, lnk mutants were assessed for susceptibility to a virulent oomycete pathogen, Hyaloperonospora arabidopsidis (Hpa). Interestingly, lnk mutants supported less disease development, suggesting a role of the wild-type LNK proteins in the enabling of pathogen colonization or in repressing host defenses. To confirm that each of the phenotypes described were a consequence of lnk1 and lnk2 mutations, wild type LNK1 and LNK2 were introduced into lnk1 lnk2 plants and examined. For most phenotypes, genetic complementation and thus restoration of a WT phenotype was observed. However, differences were uncovered in the ability of LNK genes to rescue the phenotypes, indicating specialization of function. The interaction of LNK2 with SON1 suggests that SON1-dependent ubiquitination and proteasomal degradation may control LNK2 abundance. I show by a cell free protein degradation assays that proteasome-based degradation of LNK2 as well as LNK1 is possible. Data showed that SON1 binds to ASK1 in-planta suggest the existence an SCFSON1 complex that targets LNK2 for polyubiquitination and its subsequent degradation by the proteasome. The data presented in this dissertation indicates a role for LNKs in flowering and plant defense and also suggest that proteasome-base regulation of LNK turnover may be utilized to regulate LNK protein abundance and proper maintenance of the circadian clock.

Alba family proteins are highly conserved in all domains of life. They are involved in RNA metabolism in Archae and Eucarya, while they are involved in the chromatin organisation in Crenarchaea. In animals, ALBA proteins were identified to associate with RNase P/MRP subunits. The objective of my thesis was the characterization of ALBA family proteins in a model plant Arabidopsis thaliana. The Arabidopsis genome contains six genes with close homology, three from Rpp20-like subfamily and three of Rpp25-like subfamily. Here I present the localization of GFP-fused proteins in Arabidopsis stable lines harbouring constructs cloned by Gateway® Technology. ALBA proteins were localized in the cytoplasm and undefined particles in root differentiation zone and in mature pollen. The characterization of the respective T-DNA insertion lines did not reveal significant phenotype defects in growth and development of sporophyte and gametophyte in comparison to Columbia-0 plants, probably because of likely functional redundancy od the paralogs. Expression profiles and localization of ALBA proteins suggest their possible role in differentiation and dehydration stress response in Oryza. They were also observed to associate with repressed mRNA transcripts in storage EPP particles. Collectively, I propose the likely role...

Identification et caractérisation de la première N-acylphosphatidyléthanolamine synthase chez A. thaliana. Les N-acylphosphatidyléthanolamines (NAPE) sont des phospholipides complexes peu abondants au sein des membranes biologiques mais largement répandues dans différents organismes. Outre ses fonctions de stabilisation des membranes ce lipide est davantage connu pour être le précurseur des N-acyléthanolamines (NAE) qui sont impliquées dans de très nombreuses voies de signalisation chez les plantes (lors de la germination, du développement racinaire, de l’induction de gène de défense, etc.) comme chez les animaux (apoptose, ligand des récepteurs endocannabinoïdes, notion de satiété, etc.). Au début de ma thèse, les gènes codant pour les enzymes impliquées dans les différentes étapes de la voie métabolique des NAE (e.g NAPE-PLD, FAAH1 et 2) ont été caractérisées exceptées le ou les gène(s) codant pour l’enzyme catalysant la synthèse de NAPE précurseurs de ces lipides. Une étude bioinformatique a permis d’identifier de nouvelles séquences codantes pour des acyltransférases putatives chez A. thaliana dont celle du gène At1g78690. La caractérisation fonctionnelle de cette enzyme a été déterminée après son expression hétérologue chez E.coli sur fractions membranaires et protéines purifiées. Puis le profil d’expression génique, la localisation cellulaire de la protéine ainsi que son activité ont été étudiés chez les plantes à partir notamment de mutants d’A. thaliana (ADN-T et « 35S »). Les résultats obtenus au cours de cette étude ont permis d’identifier et de caractériser la première NAPE synthase chez les plantes. / Discovery and characterization of an A. thaliana N-acylphosphatidylethanolamine synthase. N-acylphosphatidylethanolamine (NAPE) is a widespread, albeit minor, membrane phospholipid in various organisms. Besides its stabilizing properties to membranes bilayers, NAPE is known to be the precursor for N-acylethanolamine (NAE) synthesis. NAE have been shown to regulate a variety of physiological functions in both plants (germination, root development, gene induction, etc.) and animals (apoptosis, ligand for cannabinoid receptors, satiety properties, etc.) At the beginning of my PhD, the genes encoding the enzymes involved in the different steps of NAE metabolism were well characterized (e.g NAPE-PLD, FAAH 1 and 2), with the exception of the NAPE synthase gene(s). A bioinformatic study allowed the identification of coding sequences for putative new acyltransferases in A. thaliana, such as the At1g78690 gene. After expression in E. coli, the functional characterization of At1g78690p was carried out by analyses of the lipid content and by enzymatic assays using membrane fractions or purified proteins. The localisation of the protein and its activity were also studied in A. thaliana mutants (ADN-T and “35S”). This study shows the identification and characterization of the first NAPE-synthase in plants.