La carnitine, un acide aminé crucial pour le transfert intracellulaire des acides gras chez les animaux et les micro-organismes, est présente chez les plantes mais son mode d'implication dans le métabolisme lipidique et dans le développement reste à déterminer. Afin d'étudier le rôle biologique de la carnitine chez Arabidopsis nous avons initié une recherche bioinformatique d'enzymes susceptibles de participer à sa synthèse dans le but d'obtenir des mutants à teneur réduite en carnitine. Des serines hydroxyméthyl transférases (SHMT), des thréonines aldolases (THA) et des aldéhydes déshydrogénases (ALDH) candidates ont été identifiées. Une recherche de mutants, soit caractérisés, soit dans les collections disponibles, ainsi qu'une approche de mutagénèse par micro-ARN artificiel ont été initiées. Ces mutants ont été étudiés sur le plan de leur teneur en carnitine, en précurseur y-butyrobétaïne, et en esters de carnitine. Les enzymes THA ne semblent pas impliquées dans la synthèse de la carnitine et si un mutant faible de SHMT1 présente une réduction de sa teneur, et de la y-butyrobétaïne, l'implication de cette protéine reste à démontrer. L'étude d'un mutant perte de fonction du gène ALDH10A8, et de mutants baisse de fonction du gène ALDH10A9, et une complémentation fonctionnelle de mutants de levure, nous ont permis de montrer que les enzymes ALDH10 sont impliquées dans la voie de biosynthèse de la carnitine en permettant la synthèse de la y-butyrobétaïne. Les mutants des protéines ALDH10, présentant des teneurs réduites en carnitine et en acyl-carnitine, sont désormais disponibles comme outil du rôle de la carnitine chez Arabidopsis. / Carnitine, a crucial amino acid for the intacellular transfer of fatty acids in animals and microorganisms, is present in plants but its mode of implication in lipid metabolism and development remains to be determined. In order to investigate the biological function of carnitine in Arabidopsis, we initiated a bioinformatic search for enzymes that could be involved in its synthesis in order to obtain mutants with a reduced carnitine content. Serine hydroxymethyl transferases (SHMT), threonine aldolase (THA) and aldehyde dehydrogenase (ALDH) were identified as candidates. A search for mutants, either characterized or in available collections ans an amiRNA mutagenesis approach were carried out. In these mutants, the y-butyrobetaine as carnitine precursor, the carnitine, and carnitine esters were quantified. The THA enzymes do not appear to be involved in the carnitine synthesis and even if a weak mutant of SHMT1 has reduced contents of carnitine and y-butyrobetaine, the involvement of this protein remains to be demonstrated. A study of a knock-out mutant of the ALDH10A8 gene, of knock-down mutants of ALDH10A9 and a functional complementation of a C. albicans ALDH mutant, has confirmed the implication of ALDH10A8 and ALDH10A9 enzymes in the synthesis of y-butyrobetaine within the cartinine biosynthesis pathway. Mutants of the ALDH10 proteins, having significantly reduced carnitine and acyl-carnitine amounts, are now available as tools for studying the role of carnitine in Arabidopsis.

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No description available.

no / Isogenic mutants of Streptococcus mutans LT11, deficient in the production of the wall-associated protein antigens A and B, were generated by recombinant DNA technology. The hydrophobicity, adherence, and aggregation of the mutants were compared with those of the parent strain. These studies indicated that hydrophobicity, adherence, and saliva- or sucrose-induced aggregation were unaltered in the A- mutant but that hydrophobicity and adherence to saliva-coated hydroxylapatite were greatly reduced in the B- mutant whilst sucrose-dependent adherence and aggregation were increased. To determine whether these changes correlated with changes in the mutated gene product alone, the levels of a number of cell wall antigens were determined in each of the mutants. The loss of antigen A resulted in significantly reduced levels of wall-associated lipoteichoic acid, and loss of antigen B resulted in reductions in both antigen A and lipoteichoic acid. Data presented here thus suggest that changes in the expression of one wall antigen can have a dramatic effect on the levels of others.

No description available.

With the goal of creating faster and more efficient methods of generating unique Ac/Ds insertional mutants in a population of Fragaria vesca, various methods of seed germination, kanamycin screening, and the effects of varying nitrogen fertilization on diploid strawberry have been examined. Seed germination was improved to 42% in B5 liquid medium compared to _ on MS solid medium. Kanamycin screening during germination was most effective in liquid B5 medium as well. A readily discernable phonotypic difference between sensitive (necrotic radical) and resistant (branched roots) seedlings was observed in the B5 liquid medium and the frequency of escapes was reduced from __ on solid MS to __ in liquid B5. Although there were few phenotypic differences due to nitrogen application over the tested treatments (25-300 ppm) runner initiation was suppressed and chlorophyll was increased in the high (300 ppm) nitrogen treatment. There was limited evidence to suggest an increased rate of transposition in the high (300 ppm nitrogen) treatment level compared to those plants receiving lower levels of nitrogen. The selection efficiency and greater germination of the B5 liquid medium over MS medium would be expected to reduce the cost of screening thousands of seedlings because of the need for fewer disposables and medium transfers during the 5 week germination process. The use of B5 liquid medium, as well as treating plants with high levels of nitrogen (300 ppm), may be facilitate high throughput production of transposon tagged mutants in a population of F. vesca. / Master of Science

Selon l’Organisation Mondiale de la Santé, environ 30% de la population mondiale souffre de carence en fer. Une des stratégies proposées afin d’endiguer l’anémie due à cette carence est d’augmenter le contenu en fer et sa disponibilité dans les parties consommées des plantes, en particulier les graines. Pour répondre à ce défi, il est important de comprendre les mécanismes de stockage du fer dans la graine. Chez la plante modèle Arabidopsis thaliana, le fer est stocké au sein des vacuoles des cellules entourant les tissus vasculaires de l’embryon. Le transporteur AtVIT1 est impliqué dans l’entrée de fer au sein de ces vacuoles. Le mutant vit1-1 présente un patron de distribution du fer modifié : le fer est accumulé dans les cellules sous épidermiques abaxiales des cotylédons et les cellules corticales de la radicule. AtNRAMP3 et AtNRAMP4 fonctionnent de manière redondante permettant la sortie du fer vacuolaire lors de la germination. En conditions de carence en fer, le double mutant nramp3nramp4 présente un arrêt développemental associé à une forte chlorose dus à son incapacité à utiliser ses stocks de fer vacuolaire. Pour identifier de nouveaux gènes impliqués dans l’homéostasie du fer au sein de la graine, nous avons criblé une population de double mutant nramp3nramp4 mutagénisé à l’EMS à la recherche de suppresseurs du phénotype du double mutant sur un milieu carencé en fer. Nous avons nommés ces mutants isv pour “bypass iron storage in vacuoles”. Nous avons confirmé et classifié 29 candidats selon le patron de distribution du fer de leurs embryons : 20 présentent un patron similaire au type sauvage, 3 dont le patron est semblable à celui du mutant vit1-1 et enfin 6 ne présentent pas de colorations dans la plupart de leurs embryons. Les 3 mutants isv présentant un patron similaire à vit1-1ont été caractérisés de manière plus approfondie. Chez deux d’entre eux, nous avons démontré que des mutations dans le gène AtVIT1 sont responsables du phénotype suppresseur. Ce résultat établit un lien génétique et fonctionnel entre le stockage du fer dans les vacuoles endodermiques par AtVIT1 et sa libération par AtNRAMP3 et AtNRAMP4. Le troisième mutant isv au patron semblable à vit1-1 ne porte pas de mutations dans la séquence codante d’AtVIT1. L’identification du gène affecté apportera sans doute des informations sur la régulation de VIT1. Enfin, pour sept autres mutants isv, nous disposons actuellement des populations F2 dont l’analyse par séquençage haut débit permettra de déterminer la mutation responsable du phénotype suppresseur. / Over 30% of the world population is iron deficient (WHO resources). One strategy proposed to fight iron deficiency is to improve iron (Fe) content and availability in crops, especially in seeds. To address this challenge, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis thaliana seeds, iron is stored in the vacuoles of cells surrounding the vasculature of the embryo. The AtVIT1 transporter is involved in Fe influx into vacuoles. The vit1-1 mutant displays an altered Fe pattern: Fe is accumulated in abaxial cells of the cotyledons and radicle peripheral cells. AtNRAMP3 and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under iron deficient conditions, nramp3nramp4 double mutant development is altered as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an EMS mutagenized population of nramp3nramp4 for mutations restoring the growth of nramp3nramp4 on low Fe. We named these mutants isv for “bypass iron storage in vacuoles”. The 29 confirmed isv mutants identified by the screen have been classified according to the iron localization pattern in their embryo after Perls/DAB staining: 20 display a wild type pattern, 3 display a pattern of Fe localization similar to vit1-1 mutant and 6 do not show any staining in most embryos. The three isv mutants displaying a vit1-1 like pattern were further investigated. In two of them, mutations in the AtVIT1 gene were shown to be responsible for the suppressor phenotype. This result establishes a genetic and functional link between Fe loading in endodermal vacuoles by AtVIT1 and its retrieval by AtNRAMP3 and AtNRAMP4. The third isv mutant with a vit1-1 like iron localization pattern does not carry any mutation in AtVIT1 coding sequence. Identification of the mutated gene will likely uncover molecular mechanisms regulating VIT1 action. For seven other confirmed isv mutants, F2 populations are available. High-throughput sequencing of batched segregants from these F2 populations will allow to map and identify the mutations causing the suppression.

Genomic and proteomic investigations have yielded, and continue to produce, a large amount of information about genes and their protein products. In contrast, the evidence bearing on physiological roles of specific proteins is much more scarce. To address the functional part of biological inquiry, one would like to perturb, at will and selectively, the function of any protein of interest in vivo and to analyze the resulting phenotypic effects, thereby probing the protein’s role in a cell. Ideally, a method for doing so should be applicable both to individual gene products and to a large collection of them. Gene knockouts, a powerful tool to study gene function, have limitations in the study of development when the early phenotypes are cell- or organismal- lethal. Conditional mutants are particularly useful for analysis of genes whose functions are essential for the organism’s viability. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and shows an inactive phenotype under a different set of conditions, called nonpermissive; the latter must be still permissive for the wild type (wt) allele of a gene. Conditional mutants make possible the analysis of physiological changes that follow controlled inactivation of a gene or gene product and can be used to address the function of any gene. Temperature sensitive (ts) mutants are an important class of conditional mutants whose phenotype is similar to that of wt at lower (permissive) temperature, but show low or reduced level of activity above a certain temperature called restrictive temperature, while the wt gene shows a similar phenotype at both the temperatures. Ts mutants provide an extremely powerful tool to study gene expression in vivo and in cell culture. They provide a reversible mechanism to lower the level of a specific gene product simply by changing the temperature of growth of the organism. Ts mutants are typically generated by random mutagenesis; either by ultraviolet light, a chemical mutagen or by error-prone PCR followed by often laborious screening procedures. Therefore, they are cumbersome to make, especially in the case of organisms with long generation times. Keeping in view the importance of ts mutants in biology, Varadarajan et al. 1996, had developed an algorithm to predict ts mutants at predicted, buried sites of a globular protein from its amino acid sequence. Experimental tests of the algorithm were carried out on the CcdB toxin of Escherichia coli to further refine and improve the method (Chakshusmathi et al. 2004). Based on this result simple rules for the design of ts mutants were suggested. This thesis aims at validating and improving on these rules and to find out if ts mutants of a protein can also be generated by perturbing functionally important residues. In addition, it is currently unclear with what frequency ts mutants of a protein isolated in one organism will show a ts phenotype in a completely different organism. This thesis makes preliminary efforts to address this issue. The model system chosen to carry out these studies is a protein called Gal4, which is a yeast transcriptional activator. This protein is biologically relevant as it has been used for ectopic gene expression in diverse organisms including yeast, fruitflies, zebrafish, mice and frogs (Ornitz et al. 1991; Brand and Perrimon 1993; Rahner et al. 1996; Andrulis et al. 1998; Scheer and Camnos-Ortega 1999; Hartley et al. 2002). The introductory chapter (Chapter 1) discusses the importance of ts mutants and our understanding and progress in this field so far, relevant for the work reported in this thesis. Chapter 2 describes generation of ts mutants of Gal4 in yeast. Full length Gal4 (fGal4) is an 881-aa protein. To simplify the construction of ts Gal4, we have designed a functional truncated Gal4 (miniGal4 or mGal4) of 197 residues. Five residues (9, 10, 15, 18 and 23) of the Gal4 DNA binding domain, which are in close contact with the DNA, were randomized in mGal4. Based on average hydrophobicity and hydrophobic moment, 68, 69, 70, 71, and 80 are the only residues in the region 1-150 that are predicted to be buried at the 90% confidence level. Of these five sites, residues 68, 69 and 70 were chosen for mutagenesis. At these three sites, four stereochemically diverse substitutions (Lys, Ser, Ala and Trp) were made. In a separate set of experiments each predicted, buried residues were also individually randomized in both mini and in full length Gal4 (fGal4). In all cases, we have been successful in isolating ts mutants in more than one position. At both permissive and restrictive temperatures, the activity of the Gal4 ts mutants is substantially lower than the wt. However, at the restrictive temperature, the activity of the ts Gal4 is lowered below the threshold required for reporter gene expression. This view of how ts mutants function is quite different from the general notion that the ts and wt behave similarly at permissive temperatures. Chapter 3 deals with transferability of two of the ts constructs mutated at DNA binding residues (R15W and K23P) to Drosophila. Two fGal4 encoding DNA fragments carrying the mutations were cloned into P element vectors under control of Elav and GMR promoters and several transgenic Drosophila lines were generated. These were crossed to various UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. We show that both of these yeast ts mutants also show a ts phenotype in Drosophila. We have compared our ts Gal4 system with a popularly used system (TARGET) (McGuire et al. 2003) used for conditional gene expression in Drosophila. Our ts Gal4 mutants appear to provide tighter control at the restrictive temperature and a more uniform and rapid induction of gene expression upon shifting from the restrictive to the permissive temperature than the TARGET system with the promoters and the reporters we have used. Although cold sensitive (cs) mutants are often more useful than ts mutants, for reasons currently unclear, cs mutants are much more difficult to isolate than ts mutants. In Chapter 4, we have attempted to convert the ts phenotypes observed with Gal4 mutants in Drosophila and CcdB mutants in E. coli (Chakshusmathi et al. 2004) to cs phenotypes by increasing the expression level of these mutant proteins selectively at higher temperature. Several ts mutants of CcdB have been previously reported (Chakshusmathi et al. 2004). For converting the ts phenotype observed by E. coli toxin CcdB mutants (Chakshusmathi et al. 2004) to a cs phenotype, the arabinose inducible plasmid pBAD24CcdB and its mutant derivatives were used. By inducing expression of the mutant protein at higher temperature with arabinose, while keeping the basal level of expression without arabinose at lower temperature, we have been able to show cold sensitive behavior by these CcdB ts mutants in E. coli. For producing a cs phenotype with Gal4 mutants in Drosophila, we have used a P element vector where the GMR element is placed in-between hsp70 binding sites. This driver results in enhanced expression of downstream genes at 30 relative to 18°C because of the presence of the hsp elements (Kramer and Staveley 2003). Ts mutants at DNA binding and buried residues of fGal4 were cloned into this vector and several transgenic lines for each construct were obtained. The Gal4 mutants at exposed DNA binding residues but not at buried residues show a cs phonotype when they were crossed to various UAS-reporters lines. The buried residue mutants are likely to be destabilized and their degradation pathway might differ in yeast and in Drosophila. Because of this, these mutants might not be showing the desired cs phenotype in Drosophila. Although mGal4 and fGal4 have very similar activities in yeast, it was necessary to examine if they also had identical activities in Drosophila. Determining their relative activities in Drosophila is the aim of Chapter 5. To this end, mGal4 was cloned into P element vectors under control of hsp70 or GMRhs promoters and transgenic flies were generated. The transgenic lines were crossed to various UAS-reporters and reporter gene activities in the progeny were characterized. Although mGal4 and fGal4 showed similar activity in yeast, in Drosophila for reasons that are currently unclear, mGal4 was considerably less active than fGal4. As some of the fGal4 mutants showed a cs phenotype under GMRhs driver as shown in the earlier chapter (Chapter 4), several ts mutants of mGal4 in yeast in buried and as well as at the DNA binding residues were transferred to Drosophila under hs and GMRhs promoter. The transgenic lines obtained were tested for cold sensitivity by crossing with various UAS-reporter lines. However, in all cases mutant mGal4 showed an inactive phenotype in Drosophila. We suggest that this is because the intrinsic activity of these mGal4 mutants is substantially weaker than wt mGal4 even at permissive temperature in yeast. The further lowering of activity in Drosophila pushes the activity below the threshold required for reporter gene expression resulting in an inactive phenotype. The concluding chapter (Chapter 6) summarizes the conclusions drawn from this entire study and provides insights into possible mechanisms responsible for ts and cs phenotypes. The mutant phenotypes of Gal4 in yeast and in Drosophila suggest that ts phenotypes appear to result from a threshold effect. Such mutations lower the activity and/or level of the protein relative to the wt at all temperatures. Since maximal stability temperatures are rarely in excess of room temperature, with an increase in temperature, the activity of an already marginally active mutant can fall below the threshold required for function resulting in a temperature sensitive phenotype. The strategies we used for producing ts mutants have several advantages over standard approaches of generating ts alleles by random mutagenesis. We anticipate that conclusions of this study would be useful for generation of ts mutants of other globular proteins in diverse organisms. We also show that exposed, functional residues involved in protein: ligand or protein: protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms. In addition, the active site mutants of fGal4 in Drosophila, which show ts and cs phenotypes depending on the Drosophila promoter chosen for expression, can be used for conditional and reversible expression of a number of other genes using the Gal4-UAS system (Brand and Perrimon 1993).

Pseudomonas aeruginosa 47T2 i P. aeruginosa 42A2, van ser aïllats a partir sols i aigües, respectivament, contaminades amb residus oliosos. Ambdues soques van ser seleccionades per la seva capacitat per sintetitzar compostos hidrofòbics, com polihidroxialcanoats (PHAs), ramnolípids (Rhls), o estòlids. Els PHAs són polièsters biodegradables i biocompatibles de gran interès biotecnològic per ser una bona alternativa als plàstics petroquímics. A més de produir PHAs, P. aeruginosa 47T2 és capaç de produir Rhls, compostos glicolípids coneguts com surfactants microbians que presenten activitats antimicrobiona i/o antifúngica. En canvi, P. aeruginosa 42A2 no produeix Rhls, però si és capaç de convertir els olis residuals en mono-, di- o tri-hidroxi àcids grassos i més tard polimeritzar-los en estòlids. Els estòlids són biopoliesters són coneguts per ser bons emulsificadors amb interessants aplicacions industrials. Donades les diferències metabòliques a nivell lipídic detectades entre ambdues soques, s’ha estudiat el seu sistema lipolític comparant-lo amb la soca tipus Pseudomonas aeruginosa PA01. Assajos SDS-Page i zimogrames, tant dels extractes cel.lulars com dels sobrenedants de les soques, indiquen l’existència d’un sistema lipolític complex constituït per diversos enzims, però la seva caracterització bioquímica no denota diferències significatives a nivell de preferència de substrat, temperatura i pH entre les tres soques. Es decideix doncs no continuar estudiant la dotació enzimàtica, que no ha estat determinant per saber a què es deuen les diferències en el metabolisme lipídic, i s’opta per estudiar productes lipídics d’interès d’aquestes soques. Prenent els productes lipídics comuns i diferents, i tenint en compte que comparteixen intermediaris comuns a les seves rutes biosintètiques, s’ha decidit obtenir mutants negatius per la producció de PHA per tal d’estudiar el flux de carbonis i poder així dilucidar part de les rutes metabòliques que generen determinats productes. Aquests mutants es generen per ambdues soques P. aeruginosa 47T28AD i P. aeruginosa 42A28AD. Desprès d’haver provat diferents estratègies de mutagènesi aleatòria sense èxit (irradiació controlada amb llum UV i transposició), els mutants s’obtenen a través de mutagènesi dirigida amb la qual es força la recombinació i per tant la mutació dels gens que codifiquen per l’enzim PHA sintasa, mitjançant el plasmidi suïcida pEX100Tlink. S’analitzen els mutants obtinguts per microscòpia electrònica de transmissió (MET) i la producció de PHAs s’analitza per ressonància magnètica nuclear (RMN). Ambdós processos permeten determinar que els potencials mutants no produeixen PHAs i per tal de confirmar-ho s’extreu i quantifica el PHA observant que la producció de PHAs desapareix en els mutants. Una cop obtinguts els mutants cal veure si aquesta mutació afecta a la producció d’altres metabòlits. En la soca P. aeruginosa 47T2, s’estudien els Rhls produïts a partir d’olis residuals, tant a nivell de quantitat de producció com de qualitat del producte. Es quantifiquen per gravimetria i HPLC-MS i es caracteritzen a nivell fisicoquímic (determinació de CMCs, diagrames ternaris de fases, capacitat emulsionant). Si bé la producció de PHAs desapareix en els mutants seleccionats, sembla que la producció de Rhls augmenta lleugerament respecte a la soca parental, però no de manera proporcional a la desaparició de PHAs. Per altra banda la caracterització dels biotensioactius tampoc mostra diferències significatives entre els productes obtinguts a partir del mutant i de la soca parental, però si és interessant per les possibilitats a nivell d’aplicacions biotecnològiques dels Rhls. Pel que fa a la soca P. aeruginosa 42A2, els mutants serviran per fer el mateix estudi quantitatiu i qualitatiu d’un altre producte, els estòlids, però això forma part d’un altre estudi del grup. En la tesi el que s’ha quantificat són els derivats hidroxilats (mono- i di-hidroxilats) a partir dels quals es formen els estòlids, que també tenen propietats tensioactives i antimicrobianes i/o antifúngiques. / "Obtaining and characterization of PHA-negative mutants on Pseudomonas strains for the production of interesting industrial compounds" Pseudomonas aeruginosa 47T2 and P. aeruginosa 42A2 were isolated from oil wastes contaminated environment, soil and water respectively. Both strains were selected for their capacity to synthesize hydrophobic compounds such as polyhydroxyalkanoates (PHAs), rhamnolipids (Rhls), or estolides. PHAs are biodegradable and biocompatible polyesters with a high biotechnological interest for being a good alternative to the petrochemical plastics. In addition, P. aeruginosa 47T2 is able to produce rhamnolipids, glycolipid compounds well-known as microbial surfactants that present antimicrobial and/or antifungal activities. Although P. aeruginosa 42A2 is not producing Rhls, it is capable to convert waste oils into mono, di or –tri hydroxyfatty acids and later polymerize these compounds into estolides. Estolides are biopolyesters that are known for being good emulsifiers with interesting industrial applications. The aim of this thesis was to compare the differences in the lipid metabolism of the selected strain using P. aeruginosa PA01 as a type strain. SDS-page assays and zimmograms of cellular extracts and supernatants, revealed a complex lipolytic system constituted by different enzymes with no relevant differences regarding substrate affinity, temperature or pH. Therefore, the next step was the study of the different lipidic products. Considering that on PHAs, Rhls and estolides biosynthesis, the different involved metabolic pathways are competing for the same substrates, PHA-negative mutants for P. aeruginosa 42A2 and P. aeruginosa 47T2 were constructed by site-directed mutagenesis in order to elucidate the carbon flows. PHA accumulation, in both PHA-negative mutants, was evaluated by means of transmission electron microscopy (TEM) and nuclear magnetic resonance analysis (NMR), confirming thus the mutant strains were handicapped for PHA production. Consequently, rhamnolipids production in P. aeruginosa 47T2 and its PHA-negative mutant was gravimetrically quantified and showed a slightly increase of the Rhls production in the PHA-negative mutant (P. aeruginosa 47T2 4AD). Furthermore, the biopolymers were characterized by HPLC-MS and their physicochemical properties evaluated by determining the critical micelle concentration (CMC), the surfactant capacity and constructing the ternary phase diagrams. The biotransformation of oleic acid into hydroxyfatty acids was followed and quantified in P. aeruginosa 42A2 and its PHA-negative mutant (P. aeruginosa 42A2 4AD) by gas chromatography (GC).

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.