Histone H1, or linker histone, are unique histones that bind to the nucleosome to facilitate higher order chromatin structure. The linker histones, when compared to the core histones that make up the nucleosome, are poorly understood especially in plants. Linker histones are vital for plant development as well as for cell cycle regulation, sharing many qualities with animal linker histones. In this report, the first two parts introduce the current literature of H1, including result from non-plant systems, and the third section is a research proposal describing a research project to elucidate the roles of linker histones on the regulation of FLOWERING LOCUS C (FLC) in Arabidopsis thaliana. / text

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Peroxisomes are organelles originating from the endoplasmic reticulum. Peroxisome biogenesis requires multiple peroxins, including PEX19, a prenylated protein that helps deliver peroxisomal membrane proteins in yeast and mammals. Arabidopsis thaliana PEX19 is encoded by two isogenes, PEX19A and PEX19B. I demonstrate that pex19A and pex19B insertional mutants lack obvious abberant physiological phenotypes. I provide evidence that pex19A pex19B double mutants are inviable, that PEX19B is more abundant than PEX19A in young seedlings, that Arabidopsis PEX19 is farnesylated in vivo, and that YFP-PEX19 predominantly associates with what appears to be a subcellular membrane regardless of its prenylation state. I show that farnesyltransferase mutants apparently contain only non-prenylated PEX19 and lack phenotypes that would indicate inefficient peroxisome activity. My analysis of PEX19 suggests that PEX19 prenylation is dispensable for peroxisome biogenesis, and has generated tools for future studies of the earliest steps in peroxisome biogenesis in plants.

Housekeeping enzymes are vital to the metabolism of all plant cells. Two such enzymes adenosine kinase (ADK) and S-adenosylhomocysteine (SAH) hydrolase share a similar function: both sustain S-adenosylmethionine-dependent methylation reactions by removing inhibitory by-products. SAH hydrolase breaks down SAH which is a competitive inhibitor of all methyltransferase activities. ADK phosphorylates the Ado produced by SAH hydrolase and in doing so drives this reversible reaction in the hydrolysis direction. By catalyzing the phosphorylation of Ado into adenosine monophosphate, ADK not only prevents SAH from re-forming but also initiates the recycling of Ado into adenylate nucleotides and cofactors. This thesis documents two distinct research topics related to methyl recycling in plants. The first goal was to identify ADK-deficient mutants to establish the contribution of this enzyme activity to adenosine salvage. The second goal was to determine the subcellular localization of ADK and SAH hydrolase in Arabidopsis thaliana (Columbia). T-DNA insertion lines for highly similar ADK isoforms (ADK1 and ADK2) and silencing lines of overall ADK activity (sADK) were compared to WT Arabidopsis to identify phenotypic abnormalities associated with ADK deficiency. In addition to following their growth, microscopic analysis was performed on the sADK lines. While removal of either ADK1 or ADK2 had no phenotypic effect, lowering ADK levels to 6-20% that of WT lead to several changes including small, wavy rosette leaves, delayed leaf senescence, decreased internode length, reduced branching, clustered inflorescences and lack of petal abscission and silique dehiscence. Further analysis linked the abnormal phenotype to increased levels of hypomethylation throughout the plant (K Engel unpublished data), as was expected; however higher levels of active cytokinin were also observed. Thus ADK appears to be integral in regulating cytokinin levels as well as recycling methylation intermediates. To investigate the relationship between the subcellular localization of SAH production and its metabolism, immunogold labelling was performed on leaf and meristematic tissues of Arabidopsis using antibodies specific for either ADK or SAH hydrolase. As well, ???-glucuronidase and green fluorescent protein translational fusions of each enzyme were examined (S. Lee unpublished data). Results of both the immunogold labelling and fusion lines revealed that all ADK and SAH hydrolase isoforms localize to the cytosol, chloroplast and nucleus. Further analysis of purified chloroplasts has given varying results regarding the targeting of these enzymes to the organelle, and further research will be required before ADK and SAH hydrolase can be conclusively localized to the chloroplast.

Chloroplasts are photosynthetic organelles in plant and algal cells that capture sunlight energy to form energy-rich molecules that are the basis for almost all life. Chloroplast development requires more than 3000 different proteins, most of which are encoded by nuclear DNA. Thus, chloroplasts must import most of their proteins from the cytosol. They are surrounded by a double membrane called the envelope. Embedded in the envelope are the TOC and TIC complexes (translocon at the outer and inner envelope membrane of the chloroplast, respectively), which mediate protein import into the organelle. Several components of the TOC and TIC complexes have been identified. One example is the receptor Toc159, which in the model plant Arabidopsis thaliana has four isoforms: atToc159, atToc132, atToc120 and atToc90. It is known that atToc159 supports accumulation of photosynthetic proteins, while atToc132 and atToc120 support the import of non-photosynthetic, housekeeping proteins. However, the role of atToc90 remains uncertain. I investigated the function of atToc90 genetically by studying a series of Arabidopsis toc90 double and triple mutants, and by overexpressing atToc90 in mutants lacking other receptor isoforms. This work suggested limited functional redundancy between atToc90 and other TOC receptors (most notably, atToc159). By tagging TOC receptors known to act in each of the photosynthetic and non-photosynthetic import pathways, I was able to purify different TOC complexes from transgenic plants using tandem affinity purification (TAP). This indicated that atToc90 is present promiscuously in both atToc159- and atToc132/120-containing TOC complexes. Publicly available Affymetrix microarray data suggested a role for atToc90 during senescence. Thus, I investigated whether toc90 knockout mutants display any differences from wild type regarding leaf senescence. Indeed, some defects were observed, suggesting a role for atToc90 in the biochemical changes that occur in chloroplasts during leaf senescence.

Ponts entre les séquences d'acides nucléiques et les protéines, les ribosomes sont des composants essentiels des cellules vivantes. Composé d'ARN et de protéines ribosomiques, ils sont transportés, durant leurs biogenèses, du nucléole au cytoplasme, où ils traduisent les ARN messagers (ARNm) en protéines. Ces dernières années,  il a été montré que nombre de protéines ribosomiques étaient impliquées dans le développement d'Arabidopsis en intervenant sur la division et l'élongation cellulaire. L'impact d'un défaut de biogenèse des ribosomes sur le développement pourrait être expliqué par un effet dose, par une spécificité des ribosomes pour leur ARNm cibles ou par la multifonctionnalité de protéines ribosomiques. Les résultats obtenus montrent que REBELOTE (RBL), l'un des deux homologues chez Arabidopsis de la protéine NOC2p de levure, intervient probablement durant la biogenèse des ribosomes. Des mutations dans le gène RBL causent une gamme de phénotype de la létalité embryonnaire aux défauts de croissance (réduction de la taille de la plante, altération de la forme des feuilles...). Afin de mieux comprendre les processus contrôlés par RBL, la fonction ribosomique de RBL a été étudiée et ses interacteurs protéiques recherchés. Nous nous sommes ensuite focalisé sur les effets des mutations rbl sur la division et l'élongation cellulaire. Ce travail montre que les défauts observés aux niveaux moléculaire et cellulaire peuvent expliquer les retards de croissance des mutants rbl. / Bridges between nucleic acids sequences and proteins, ribosomes are central components and the “auletes” of living cells.  Composed of ribosomal proteins and RNA, they move during their biogenesis from the nucleolus to the cytoplasm, where they translate RNA messengers into proteins. In the past years, some mutants of ribosomal-biogenesis-related proteins have shown the importance of these proteins during cell division and Arabidopsis development. The impact of ribosomal defects on development could be explained by dose effect (which could be important for cell fitness), specificity of ribosomes for some mRNA or multifunctional ribosomal proteins (Mary E. Byrne, 2009). Here I present our work on REBELOTE (RBL), one of the two Arabidopsis homologs of the yeast NOC2 protein, which act during the ribosomal 60S subunit biogenesis. Mutations in REBELOTE gene cause a range of phenotypes, from embryo lethality to growth defects (reduced plant size, altered leaf shape…). To have a better understanding of RBL-controlled processes, we first analyzed the ribosomal function of RBL, and searched for its protein partners. Our results shows that RBL act in two different nucleolar complexes supposed to regulate 60S ribosomal subunit biogenesis. Subsequently, we focused on the effects of rbl mutations on the cell division/elongation processes. Our work shows that defects observed at molecular and cellular levels could explain the slow down of cell divisions and growth delay in rbl mutants.

The natural genetic variation among A. thaliana populations in Catalonia was used to identify local adaptation to coastal and inland habitats. A Species Distribution Model (SMD) was created to locate multiple small stands of A. thaliana (demes). Results using 425 genome-wide SNP markers under clustering and population analysis indicate a high percentage of shared alleles among demes. Multi-year field-based reciprocal transplant experiments were designed to identify fitness trade-offs between inland and coastal demes. Progenies from these demes performed better in their local/home environments. Similar results were obtained in greenhouse common garden experiments, confirming that soil is a driving factor for local adaptation. Plants from the coastal habitat outperformed those from inland when grown together under high salinity. It is concluded that A. thaliana is locally adapted to coastal environments, and this adaptation is driven, at least in part, by the elevated salinity of coastal soils. Our results do not point to a single mechanism of salinity tolerance. AtSOS1 and AtHKT1;1 may cooperate increasing leaf Na+ and its vacuolar storage achieving better osmotic adjustment. Crossings between coastal (salt tolerant) and inland (salt sensitive) plants suggest maternal inheritance of salt tolerance. Polymorphisms in both AtHKT1;1 and AtMOT1 may be of adaptive significance because the weak alleles were only detected in coastal demes. However, all results indicate that genetic variability in AtHKT1;1 allele is not responsible for the salinity tolerance. We conclude that the weak allele of AtHKT1;1 persists and coexists with plants bearing the strong allele thanks to early flowering and better tolerance of moderate salinity. Moreover, the weak allele of AtMOT1 was more frequent and was detected nearer to the sea than the weak allele of AtHKT1;1. Results with mot1 knockout mutants under NaCl treatments indicate that loss of function of AtMOT1 may enhance tolerance to salt stress.