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Biochimie et biologie structurale appliquées à l’oenologie : étude des protéines de raisin Thaumatin-like et Chitinase / Biochemistry and structural biology for oenology : study of thaumatin-like and chitinase grape proteinsLe Bourse, Doriane 09 December 2011 (has links)
Les protéines de raisin thaumatin-like et chitinase sont d’un intérêt majeur, tant pour la recherche viticole de par leur implication dans les mécanismes de défense de la vigne, que pour la recherche en oenologie en raison de leur présence en quantité majoritaire dans le pool protéique d’un jus de raisin. La mise au point d’un protocole de quantification par chromatographie liquide et dosage immuno-enzymatique utilisant des gammes de protéines purifiées à partir de jus de raisin a permis de caractériser la diminution de la concentration de ces deux protéines au cours du procédé de vinification. Les propriétés tensioactives des deux protéines thaumatin-like et chitinase purifiées ont été évaluées, révélant que ni l’une ni l’autre ne pouvait à elle seule expliquer la formation et la stabilisation des bulles et de la mousse d’un Champagne. Une étude structurale de la protéine thaumatin-like VVTL1 a ensuite été menée dans l’optique de mieux comprendre les mécanismes chimiques, biologiques et physiques dans lesquels elle peut être impliquée. Une structure de VVTL1 a été modélisée par homologie et l’analyse de ses modes normaux a permis de révéler un mécanisme de type mâchoire autour d’une cavité acide, site potentiel de l’activité enzymatique de la protéine. Un feuillet beta en épingle à cheveux isolé dans la structure s’est révélé être très conservé et absolument spécifique à la superfamille des protéines thaumatin-like, ouvrant peut-être la voie vers l’élucidation complète du rôle biologique de ces protéines. Dans une seconde approche, la détermination de la structure d’un peptide sélectionné dans la séquence de VVTL1 par modélisation sous contraintes RMN a posé les bases d’une étude modèle de l’adsorption des protéines à la surface de la bentonite, argile utilisée pour la clarification des vins. / Grape proteins thaumatin-like and chitinase are of major interest, as much by the vine defense mechanisms they are involved in as by their dominance over the grape juice protein pool. Liquid chromatography and immunoassays allowed both proteins to be quantified in grape juice and Champagne, showing that their concentration decreases through winemaking. The involvement of these proteins in gas/liquid interfaces was also studied on the purified fractions from grape juice previously made for quantification standards. Results clearly indicated that neither thaumatin-like nor chitinase could alone explain bubble formation and foam stabilization in Champagne. A first study of the three-dimensional structure of the main thaumatin-like protein VVTL1 using homology molecular modeling was then achieved and normal modes analysis was performed on the VVTL1 model. It revealed a jaw-like mechanism opening and closing an acidic cleft assessed to be the enzymatic binding site. An isolated beta hairpin turned out to be highly conserved and specific to the thaumatin-like superfamilly. This domain could provide a first clue to unravel the mystery of the protein biological activity in the field of plant-pathogen interactions. A second approach was set up for the structure determination of a VVTL1 peptide using molecular modeling under NMR restraints. It could lead to a model study of protein adsorption on bentonite, a clay used for wine clarification.
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Expression spatio-temporelle de deux protéines PR du grain de raisin - dégradation au cours de l'infection par Botrytis cinerea - / Spatio-temporal study of two PR-proteins of grape berries -degradation during infection by Botrytis cinerea-Colas, Steven 28 November 2012 (has links)
L'infection des baies de raisin par le champignon Botrytis cinerea, responsable de la pourriture grise, est fréquente et occasionne des dégâts importants. Pourtant, il semble que la baie dispose de moyens de défense parmi lesquels des protéines PR "Pathogenesis-Related". Chez le Pinot Noir, une chitinase (CHI4D) et une thaumatin-like (TL3) s'accumulent naturellement en grande quantité à partir de la véraison et présentent une activité antifongique contre B. cinerea in vitro. L'objectif de ce travail est de comprendre comment B. cinerea peut se développer sur des baies censées disposer de défenses suffisantes. Pour cela, l'expression spatio-temporelle des ARNm et des protéines CHI4D et TL3 a été suivie respectivement par hybridation in situ et immunohistolocalisation dans les baies, au cours de la maturation, à la suite d'un stress abiotique (UV-C) ou d'un stress biotique (B. cinerea). Dans des baies avant véraison (vertes), n'exprimant naturellement que très faiblement CHI4D et TL3, les ARNm et les protéines s'accumulent en grande quantité après application d'un stress abiotique (UV-C) ou biotique (infection artificielle par B. cinerea). Les protéines CHI4D et TL3 sont localisées au niveau des faisceaux conducteurs ainsi qu'au niveau des tissus proches des sites d'exposition aux UV-C (exocarpe) ou au niveau des sites d'inoculation de B. cinerea, suggérant qu'elles sont impliquées dans la défense de la baie avant véraison. Après véraison, les ARNm et les protéines sont naturellement accumulés au niveau de l'exocarpe et des faisceaux conducteurs qui correspondent à des sites potentiels d'entrée ou de propagation des agents pathogènes. Alors que l'application d'un stress UV-C sur ces baies ne provoque qu'un effet mineur sur l'expression de CHI4D et de TL3, au cours de l'infection par B. cinerea, les quantités de transcrits et de protéines diminuent. A un stade précoce d'infection, la diminution de la quantité des deux protéines est observée en avant du front de propagation du champignon, suggérant une dégradation par des protéases sécrétées par B. cinerea. A un stade d'infection plus avancé, cette diminution s'étend à l'ensemble de la baie. La production hétérologue des protéines CHI4D et TL3 nous a permis de confirmer que CHI4D pouvait être dégradée par des protéases aspartiques sécrétées par B. cinerea. La dégradation de TL3 n'a pas pu être reproduite in vitro. Des tests antigerminatifs effectués in vitro avec les protéines hétérologues n'ont pas permis de mettre en évidence d'effet antifongique malgré la présence d'une activité chitinase pour CHI4D et β-1,3,-glucanase pour TL3. Il est donc possible que ces protéines possèdent des fonctions autres que celles impliquées dans la défense. / Grape berries infection by the phytopathogenic fungus Botrytis cinerea, the causal agent of gray mold, is quite common and causes significant damage. However, it seems that berries have a mechanism of defense, among which are pathogenesis related proteins. In Pinot Noir grape berries, a chitinase (CHI4D) and a thaumatin-like (TL3) protein naturally accrue in large amounts from véraison and show in vitro an antifungal effect against B. cinerea. The aim of this work was to understand how B. cinerea can develop on grape berries that seem to have sufficient defense mechanisms. To do so, the spatio-temporal expression of CHI4D and TL3 mRNAs and proteins in berries was studied respectively by in situ hybridization and immunohistolocalization during maturation, after an abiotic stress (UV-C) or a biotic stress (B. cinerea). Before véraison (green berries) while the expression of CHI4D and TL3 is naturally low, mRNAs and proteins have accumulated in large amounts in berries after UV-C exposition or artificial infection with B. cinerea. CHI4D and TL3 proteins have accumulated around vascular bundles as well as near the sites of UV-C exposition (exocarp) or B. cinerea inoculation, suggesting that before véraison these proteins could be involved in the berry defense. After veraison, mRNAs and proteins naturally accumulate in the exocarp and around vascular bundles that correspond to potential sites of penetration or propagation of pathogenic agents. While the application of UV-C stress on these berries causes only a minor effect on the expression of CHI4D and TL3, during infection by B. cinerea, the amounts of mRNA and proteins decreased. At an early stage of infection, the less amounts of both proteins were observed around the fungus propagation area, suggesting that these proteins could be degraded by B. cinerea secreted proteases. At a more advanced stage of infection, the decrease extended to the entire berry.Production of heterologous CHI4D and TL3 proteins allowed us to confirm that CHI4D could be degraded by aspartic proteases secreted by B. cinerea whereas no degradation of TL3 could be observed in vitro. Both heterologous proteins showed no antifungal effect while a chitinase and a β-1,3-glucanase activities were observed respectively for CHI4D and TL3. It is therefore possible that these proteins have other functions than those involved in the defense.
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Functional analysis of proteins in the conifer ovular secretionCoulter, Andrea Elizabeth 31 August 2020 (has links)
Almost all conifer ovules produce a liquid secretion as part of reproduction. This secretion, termed an ovular secretion, is produced during ovule receptivity and is involved in pollen capture and transport. Historically, examinations of the ovular secretion have focused on how they are part of pollination mechanisms. As a result, the chemical composition of the ovular secretion has not been examined systematically. Investigations into the constituents of the ovular secretion were limited to analyses for simple water soluble compounds such as sugars, minerals, amino acids and organic acids. More recently, the protein component of the secretion has been investigated using mass spectrometry-based proteomics. Proteins involved in processes such as carbohydrate modification, proteolysis, and defence have been identified in conifer ovular secretions. This biochemical complexity suggests a broader view of the function of the ovular secretion is warranted. However, protein identifications only provide putative information on function. Functional characterization of these proteins is needed in order to fully understand how they contribute to ovular secretion function. The research outlined in this dissertation describes the first functional characterizations of proteins found in conifer ovular secretions. Three proteins - invertase, chitinase, and thaumatin-like protein - were characterized in the ovular secretions of Douglas-fir (Pseudotsuga menziesii) and hybrid yew (Taxus × media). The Douglas-fir ovular secretion is capable of converting sucrose to glucose and fructose, confirming that invertases present in the secretion are functional. The invertase activity was maximal at pH 4.0. Activity was 77% of maximal at pH 4.5, the physiological pH. This indicates that post-secretory hydrolysis of sucrose occurs in situ in the Douglas-fir ovular secretion. Invertases in the ovular secretion are likely involved in controlling the movement of carbohydrates to developing pollen and could facilitate pollen selection. Chitinases present in the Douglas-fir ovular secretion are functional at physiological conditions. All three modes of chitinolytic activity, i.e. endochitinase, chitobiosidase and β-N-acetylglucosaminidase, were detected at physiological pH. β-N-acetylglucosaminidase activity was 80 % of maximal at physiological pH. Chitinases are pathogenesis-related proteins capable of hydrolysing chitin in fungal cell walls. These results suggest the ovular secretion is capable of defending the ovule against infection by phytopathogens. Thaumatin-like protein was immunolocalized to the cell wall and amyloplasts in Douglas-fir and yew nucellar tissue in a pattern consistent with a defensive role. It was also localized to the cell wall of fungal spores and germinating hyphae that were present in the micropyle of a yew ovule. These results provide additional evidence for an antifungal role for the ovular secretion. Functioning enzymes involved in pollen-ovule interactions and ovule defence are present in the conifer ovular secretion. The ovular secretion has functions beyond pollen capture. A revised functional model for the conifer ovular secretion is proposed. / Graduate / 2021-08-17
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