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No description available.

Protein import into chloroplasts is mediated by translocons of the outer and inner envelope membranes, termed TOC and TIC, respectively. To investigate the in vivo roles of three TIC components, Tic40, Ticl 10 and Hsp93, Arabidopsis null mutants for each were identified and characterized. The tic40, ticl 10 and hsp93-Vmutants displayed distinct chlorotic phenotypes and aberrant chloroplast ultrastructure. Furthermore, in vitro protein import assays using mutant chloroplasts revealed that each mutant has a general protein import defect. Genetic interaction studies performed with these mutants also revealed that the proteins may cooperate functionally, supporting their proposed cooperation in a putative protein import motor. Two approaches were taken to further investigate the role of Tic40 in vivo. Firstly, a genetic screen was performed for suppressor of tic40 (stic) mutants that alleviate the chlorotic phenotype of tic40. Several stic mutants were identified, and their analysis revealed that they were the result of semi-dominant and recessive mutations. All the mutants analysed showed a slight improvement in thylakoid development, and an increase in leaf cell number and density, consistent with the possibility that the mutants display increased import efficiency. Mapping may reveal that the affected genes encode functional partners of Tic40, such as Ticl 10 and Hsp93. Secondly, the significance of homology between Tic40 and the human co-chaperone, Hip, was investigated. Tic40 deletion and Tic40:Hip fusion constructs were generated, and transformed into tic40 to assess for complementation. Interestingly, two deletion constructs that failed to complement tic40 had a dominant-negative effect, causing a phenotype more severe than that of tic40. The corresponding proteins are proposed to associate with the TIC and interfere with its activity. Remarkably, Tic40:Hip fusions revealed that although Tic40 and Hip are not functionally equivalent, the extreme C-terminus of Hip can fully replace the homologous region of Tic40. This finding provides the first experimental evidence indicating that Tic40 may function as a co-chaperone.

With the increasing evidence of climate change in the coming decades, adaptive mechanisms present in nature may permit crop survival and growth on marginal or saline soils and is considered an important area of future research. Some subspecies of Restharrow; O. repens subsp. maritima and O. reclinata have developed the remarkable ability to colonise sand dunes, shingle beaches and cliff tops. α-onocerin is a major component within the roots of Restharrow (Ononis) contributing up to 0.5% dry weight as described by Rowan and Dean (1972b). The ecological function of α-onocerin is poorly understood, with suggestions that it has waterproofing properties, potentially inhibiting the flow of sodium chloride ions into root cells, or preventing desiccation in arid environments. The fact that alpha-onocerin (a secondary plant metabolite) biosynthesis has evolved a number of times in distantly related taxa; Club mosses, Ferns and Angiosperms, argues for a relatively simple mutation from non-producing antecedents. No direct research has been reported to have investigated the biosynthetic mechanism towards α-onocerin synthesis via a squalene derived product originally characterised by Dean, and Rowan (1972a). A bi-cyclisation event of 2,3;22,23-dioxidosqualene by an oxidosqualene cyclase, may provide plants with an alternative mechanism for synthesising a range of triterpene diol products via alpha-onocerin (Dean, and Rowan, 1972a). This mechanistic possibility presented the opportunity to investigate the biosynthesis of α-onocerin using a multi-disciplinary approach. This thesis presents supporting data, that α-onocerin is a derivative of 2,3;22,23-dioxidosqualene via an oxidosqualene cyclase. Genetic markers were developed for Ononis versions of squalene cyclase, squalene epoxidase and a putative oxidosqualene cyclase. It was determined that squalene epoxidase; At4g37760 (SQE3), from A. thaliana showed the highest level of amino acid sequence conservation with the O. spinosa version. SQE3 is known to cyclise 2,3;22,23-dioxidosqualene (Rasbery et al., 2007). Based on amino acid sequence alignments and predictive protein modelling a partial putative oxidosqualene cyclase isolated from O. repens III subsp. maritima is likely to be an Ononis version of β-amyrin synthase rather than a multifunctional oxidosqualene cyclase. Further functional characterisation studies are needed. Methods were developed for analysing the transcriptome and metabolome in Restharrow which will aid future functional characterisation studies. Within O. spinosa root SQE3 was highly expressed. In contrast SQE3 was expressed at low levels in O. spinosa leaf and O. pusilla root and leaf. This data was supported by metabolomic profiling of five species of Restharrow; O. spinosa, O. repens, O. repens subsp. maritima, O. pusilla and O. rotundifolia. Triterpenes α-onocerin and 2,3;22,23-dioxidosqualene were not present in O. rotundifolia and O. pusilla. Where 2,3;22,23-dioxidosqualene was present in plant extracts, α-onocerin accumulation was also detected. O. pusilla and O. spinosa can be utilised for studying the occurrence of alpha-onocerin within plants. The data presented in this thesis provides the necessary background information providing targets for functional expression studies of squalene epoxidases and oxidosqualene cyclases from Restharrow. In summary the results in this thesis support the hypothesis proposed by Dean and Rowan (1971). There is good evidence to suggest the ability of Restharrow to cyclise alpha-onocerin, may be dependant on the availability of 2,3;22,23-dioxidosqualene as the primary precursor. This was shown in development, tissue specific, ecotype and cell free enzyme analytical chemistry assays. There was little evidence to suggest a single specific oxidosqualene cyclisation event was primarily responsible for alpha-onocerin biosynthesis. The work also presents evidence to suggest that differences in the squalene epoxdiase sequence and transcription signals may affect the plants ability to cyclise alpha-onocerin. This may have ecological implications and allow plants to adapt to their environment by providing and alternative route to biosynthesising membrane constituents via an alternative substrate specific mechanism.

Les cellules épidermiques des téguments des graines d’Arabidopsis thaliana, espèce myxospermique, libèrent un halo de mucilage polysaccharidique lors de leur imbibition. Les polysaccharides du mucilage sont produits et accumulés au cours du développement de la graine, selon un processus de différenciation déjà largement décrit (Western et al. 2006). Au laboratoire, une mutation naturelle a été mise en évidence chez l’accession Djarly, dont les graines ne libèrent pas de mucilage au cours de leur imbibition. Le clonage positionnel a démontré que le locus affecté chez Djarly code pour un inhibiteur de pectine méthylestérase (PMEI6). Les PMEIs exercent un contrôle négatif sur l’activité des pectines méthylestérases (PME), enzymes qui déméthylestérifient les homogalacturonanes, par la formation d’un complexe PME-PMEI (Di Matteo et al., 2005 ; Hothorn et al., 2004). Des études génétiques, cytologiques et biochimiques ont prouvé que PMEI6 régule la méthylestérification des homogalacturonanes du mucilage et des parois cellulaires distales des cellules épidermiques de la graine retardant la libération du mucilage séminal. L’expression de PMEI6 dépend des régulateurs de transcription GLABRA2 et MUM1. L’activité PME dans les cellules épidermiques des graines est aussi modulée par la subtilisine serine protéase AtSBT1.7, et le phénotype additif du mutant pmei6 atsbt1.7 indique que PMEI6 régule d’autres PMEs. Djarly fait partie d’un groupe de vingt accessions, dont les graines flottent à cause de modifications des propriétés du mucilage séminal. Ces accessions portent au moins dix mutations indépendantes, qui affectent au moins 4 locus différents. Cette étude nous a permis de proposer que la modification des propriétés du mucilage est impliquée dans l’adaptation à l’environnement local, permettant la dispersion à longue distance des graines par l’eau. / Upon imbibition, the myxospermous seeds of Arabidopsis thaliana, form a mucilage from hydrated polysaccharides released from the epidermal cells of the seed coat. These polysaccharides are produced and accumulated during seed development in a differentiation process that has been described in detail (Western et al. 2006). A screen of Arabidopsis accessions identified Djarly as a natural mucilage mutant affected in mucilage release on imbibition. The locus defective in Djarly was identified by map-based cloning as encoding a pectin methylesterase inhibitor (PMEI6). Theseproteinaceous inhibitors negatively control the activity of pectin methylesterases (PME), enzymes that demethylesterify HG, through the formation of a PME-PMEI complex (Di Matteo et al., 2005; Hothorn et al, 2004). Genetic, cytological and biochemical studies demonstrated that PMEI6 regulates methylesterification of homogalacturonans present in mucilage and the outer cell wall of seed coat epidermal cells. Delayed seed mucilage release in pmei6 mutants results, therefore, from the reduced level of homogalacturonan methylesterification. Expression of PMEI6 required the transcription regulators GLABRA2 and MUM1. PME activity in seed coat epidermal cells is also modulated by the subtilisin serine protease AtSBT1.7, and the additive phenotype of pmei6 atsbt1.7 mutants indicates that PMEI6 regulates different PMEs. Djarly is one of twenty accessions where seeds float due to modifications of mucilage properties. At least ten independent mutations are responsible for the mucilage modifications in these accessions, affecting at least 4 different loci. This study has led us to propose that these mucilage modifications are local adaptations that allow longdistance seed disperal on water.

Le but de cette thèse était d’identifier les mécanismes moléculaires responsables des propriétés particulières de la couche G et les propriétés mécaniques remarquables du bois de tension (BT). Trois acteurs moléculaires potentiels (des protéines à arabinogalactane avec domaine fasciclin-like (FLA), une protéine chitinase-like (CTL) et une β-galactosidase (BGAL)) ont été choisis et étudiés dans: analyse phylogénétique, analyses d'expression et caractérisation de peupliers transgéniques affectés dans l’expression de chacun de ces acteurs. La caractérisation fine de ce matériel a révélé que CTL2 et les FLA jouent un rôle dans la régulation de la cristallinité de la cellulose dans le BT. CTL2 apparaît également important dans l'organisation de la paroi cellulaire et des propriétés mécaniques des tiges. BGAL a été avant proposé pour une fonction dans modification de pectine RG-I potentiellement important pour des propriétés mécaniques de BT. Le bois de tension exhibe une activité BGAL plus élevée que dans le bois opposé. L’inhibition par RNAi de l’expression de BGAL7, spécifiquement exprimée dans le BT, n’est pas responsable à lui seul de la forte activité BGAL présente dans le BT. En contrepoint à l’étude menée sur le peuplier, nous avons également évalué la présence d’acteurs moléculaires potentiellement responsables des propriétés mécaniques du BT chez le simarouba qui développe dans leur BT des fibres ayant leurs sous-couches de la paroi intermédiaire entre la G et la S2. Des protéines à arabinogalactanes ainsi que des pectines du type RG-I sont présentes dans les fibres de BT de peuplier et de simarouba et pourraient avoir une fonction dans un mécanisme commun de génération des contraintes dans le BT. Finalement, un modèle est proposé sur le rôle présumé des différents acteurs moléculaires étudié dans la régulation des propriétés de la couche G et la génération des fortes contraintes du bois de tension. / The aim of this thesis was to approach the underlying molecular mechanisms responsible for the particular properties of the G-layer and the outstanding mechanical properties of tension wood (TW). Accordingly, three potential molecular players (fasciclin-like arabinogalactan protein (FLA), chitinase-like protein (CTL) and β-galactosidase (BGAL)) were chosen and studied through a phylogenetic analysis, expression analyses and most importantly characterization of RNAi transgenic poplars. This multilevel characterization revealed that CTL2 and FLAs have function in the regulation of cellulose crystallinity in TW. CTL2 was also shown to be important both for the cell wall organization and stem mechanical properties. BGAL was studied in a light of the previously reported modifications of RG-I pectin, potentially important for the mechanical properties of TW. Study of BGAL revealed that the enzyme has higher activity in TW than in opposite wood. BGAL7, whose gene was expressed specifically in TW, does not seem to be responsible for the higher BGAL activity in TW. In comparison to poplar, we analyzed the occurrence of molecular players potentially responsible for TW mechanical properties in simarouba, a tropical species, which develops different TW fiber. Arabinogalactan proteins and RG-I pectin potentially targeted by BGAL were localized in TW fibers both in poplar and simarouba and therefore may be involved in a common mechanism of tensile stress generation in different TW types. A model was finally proposed to elucidate a potential function of the studied molecular players in the regulation of G-layer properties and tensile stress generation.