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Etude Proteomique De La Vacuole D'arabidopsis Thaliana En Vue De L Etude proteomique de la vacuole d’Arabidopsis thaliana en vue de l’identification d’acteurs protéiques impliqués dans la détoxication du cadmium Florent Villiers To cite this version: Florent Villiers. Etude proteomique de la vacuole d’Arabidopsis thaliana en vue de l’identification d’acteurs protéiques impliqués dans la détoxication du cadmium. Biologie végétale. Université Joseph- Fourier - Grenoble I, 2008. Français. tel-00652513 HAL Id: tel-00652513 https://tel.archives-ouvertes.fr/tel-00652513 Submitted on 15 Dec 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Université Joseph Fourier – Grenoble I THESE Pour obtenir le titre de DOCTEUR DE L’UNIVERSITE JOSEPH FOURIER Discipline : Biologie Présentée par Florent VILLIERS Le 31 octobre 2008 ETUDE PROTEOMIQUE DES VACUOLES D’ ARABIDOPSIS THALIANA , EN VUE DE L’IDENTIFICATION D’ACTEURS PROTEIQUES IMPLIQUES DANS LA DETOXICATION DU CADMIUM Composition du jury : Patrick RAVANEL Président Alain PUGIN Rapporteur Michel ROSSIGNOL Rapporteur Michel JAQUINOD Invité Jacques BOURGUIGNON Directeur de thèse Co-directrice de thèse : Véronique HUGOUVIEUX Thèse préparée au sein du Laboratoire de Physiologie Cellulaire Végétale UMR CNRS 5168 – INRA 1200 – CEA – UJF CEA – GRENOBLE à mes parents, à mon épouse, Clissandre … Université Joseph Fourier – Grenoble I THESE Pour obtenir le titre de DOCTEUR DE L’UNIVERSITE JOSEPH FOURIER Discipline : Biologie Présentée par Florent VILLIERS Le 31 octobre 2008 ETUDE PROTEOMIQUE DES VACUOLES D’ ARABIDOPSIS THALIANA , EN VUE DE L’IDENTIFICATION D’ACTEURS PROTEIQUES IMPLIQUES DANS LA DETOXICATION DU CADMIUM Composition du jury : Patrick RAVANEL Président Alain PUGIN Rapporteur Michel ROSSIGNOL Rapporteur Michel JAQUINOD Invité Jacques BOURGUIGNON Directeur de thèse Co-directrice de thèse : Véronique HUGOUVIEUX Thèse préparée au sein du Laboratoire de Physiologie Cellulaire Végétale UMR CNRS 5168 – INRA 1200 – CEA – UJF CEA – GRENOBLE Remerciements A l’heure de la synthèse du travail que j’ai effectué pendant ma thèse, je souhaite remercier les personnes qui ont contribué à sa réalisation. Ce sont elles qui m’ont formé, qui m’ont conseillé, et qui m’ont soutenue ; qu’elles reçoivent ici le témoignage de ma gratitude et de mon affection. Je souhaite en premier lieu remercier le Dr Marylin Vantard, qui dirige le laboratoire de Physiologie Cellulaire Végétale qui m’a accueillit pour cette thèse. Merci infiniment à Jacques Bourguignon pour la grande qualité de son encadrement. Son expertise scientifique, sa grande rigueur, la confiance qu’il a bien voulu m’accorder et ses qualités humaines ont été primordiales à la réalisation de mon travail et au plaisir que j’y ai pris. Merci également à Véronique Hugouvieux. Son savoir-faire scientifique et pédagogique m’a permis d’exploiter les outils de la biologie moléculaire efficacement et de me former à cet aspect de la recherche. La qualité de ses conseils a fait de son encadrement une part essentielle de ma formation. Je suis aussi extrêmement reconnaissant à Michel Jacquinod pour son implication dans mon projet de thèse. Sa disponibilité et sa grande compétence m’ont permis de me familiariser avec la spectrométrie de masse. Une part importante des travaux présentés a été réalisée dans la cadre d’une collaboration avec le laboratoire d’Etude de la Dynamique des Protéomes. Je souhaite ici exprimer ma reconnaissance à Jérôme Garin, qui le dirige, et à Sylvie Kieffer-Jaquinod, Lauriane Kuhn, Maighread Gallagher et Alexandra Kraut, avec qui j’ai été plus particulièrement en interaction. Merci à tous ceux avec qui j’ai eu la chance de travailler, au laboratoire de Physiologie Cellulaire Végétale ou ailleurs. Merci en particulier à Nathalie Leonhardt, à Agnès Jourdain, à Nolwenn, Oliv’, Delph’, Emma et Hélène. Je tiens également à exprimer ma reconnaissance au Pr Alain Pugin, au Dr Michel Rossignol, et au Pr Patrick Ravanel qui m’ont fait l’honneur de juger mon travail. Je les remercie pour leurs critiques et leurs commentaires. Enfin mes plus vifs remerciements vont à ma famille. Merci à mes parents, pour leur continuel soutien et leur précieuse aide tout au long de mon cursus, en particulier lors de la rédaction de ce manuscrit. Et puis merci à toi Clissandre pour ta compréhension, pour ta douceur et pour ton appui. Tu m’as accompagné à chaque instant de ma thèse, ce travail est aussi le tiens. - 1 - Sommaire SOMMAIRE ..........................................................................................................................................................................2 INDEX DES FIGURES .............................................................................................................................................................6 INDEX DES TABLEAUX .........................................................................................................................................................9 ABREVIATIONS ..................................................................................................................................................................10 INTRODUCTION GENERALE ................................................................................................................................................12 SYNTHESE BIBLIOGRAPHIQUE..................................................................................................................................14 I - LA VACUOLE VEGETALE .....................................................................................................................................14 I.1 - Occurrence des vacuoles dans le vivant............................................................................................... 14 I.2 - Rôles de la vacuole végétale et transporteurs impliqués dans ses fonctions........................................ 16 I.2.1 - Maintien de l’homéostasie cellulaire.......................................................................................... 17 I.2.2 - Implication de la vacuole dans des fonctions métaboliques....................................................... 25 I.2.3 - Protection cellulaire.................................................................................................................... 29 II - LE STRESS CADMIUM CHEZ LES PLANTES ..........................................................................................................31 II.1 - Entrée du cadmium dans la plante ....................................................................................................... 32 II.2 - Chélation des métaux, et plus particulièrement du cadmium, dans la plante / Répartition tissulaire des ions métalliques.................................................................................................................................................. 33 II.2.1 - Transport par le système vasculaire ........................................................................................... 33 II.2.2 - Distribution du cadmium dans la plante : cas particulier des trichomes..................................... 35 II.3 - Mécanismes cellulaires de la détoxication du cadmium ...................................................................... 35 II.3.1 - Synthèse des phytochélatines ..................................................................................................... 36 II.3.2 - Compartimentation des métaux lourds dans la vacuole.............................................................. 40 III - OBJECTIFS DE LA THESE .....................................................................................................................................42 MATERIELS ET METHODES.........................................................................................................................................44 I - BIOLOGIE CELLULAIRE ......................................................................................................................................44 I.1 - Matériel Végétal .................................................................................................................................. 44 I.1.1 - Cellules en culture liquide d’ Arabidopsis thaliana .................................................................... 44 I.1.2 - Cultures in vitro .......................................................................................................................... 44 I.2 - Purification de vacuoles de cellules en culture d’ Arabidopsis thaliana ............................................... 45 I.2.1 - Préparation de protoplastes ........................................................................................................ 45 I.2.2 - Isolement de vacuoles (méthode adaptée de Frangne et al. , 2002) ............................................ 45 I.3 - Préparation de fractions enrichies en cytosol....................................................................................... 46 II - TECHNIQUES BIOCHIMIQUES ..............................................................................................................................47
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