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Proteomic Analysis of Endosperm and Peripheral Layers During Kernel Proteomic analysis of endosperm and peripheral layers during kernel development of wheat (Triticum aestivum L.) and a preliminary approach of data integration with transcriptome Ayesha Tasleem-Tahir To cite this version: Ayesha Tasleem-Tahir. Proteomic analysis of endosperm and peripheral layers during kernel develop- ment of wheat (Triticum aestivum L.) and a preliminary approach of data integration with transcrip- tome. Agricultural sciences. Université Blaise Pascal - Clermont-Ferrand II, 2012. English. NNT : 2012CLF22252. tel-00923145 HAL Id: tel-00923145 https://tel.archives-ouvertes.fr/tel-00923145 Submitted on 2 Jan 2014 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. UNIVERSITE BLAISE PASCAL UNIVERSITE D’AUVERGNE N° D.U. 2252 ANNEE 2012 ECOLE DOCTORALE DES SCIENCES DE LA VIE, SANTE, AGRONOMIE, ENVIRONNEMENT N° d’ordre 585 Thèse Présentée à l’Université Blaise Pascal Pour l’obtention du grade de DOCTEUR D’UNIVERSITE Spécialité: Physiologie et génétique moléculaire Soutenue le 4 Juillet 2012 Ayesha TASLEEM-TAHIR ______________________________________________________________________________ Analyse du protéome de l’albumen et des couches périphériques du grain de blé (Triticum aestivum L.) en développement: vers une intégration des données avec le transcriptome Proteomic analysis of endosperm and peripheral layers during kernel development of wheat (Triticum aestivum L.) and a preliminary approach of data integration with transcriptome ______________________________________________________________________________ Président : Saïd MOUZEYAR Professeur, UBP, Clermont Ferrand, France Rapporteurs : Birte SVENSSON Professeur, DTU, Lyngby, Denmark Dominique JOB Directeur de Recherche, CNRS/BAYER, Lyon, France Examinateur : Didier MARION Directeur de Recherche, INRA, Nantes, France Directeur de thèse : Gérard BRANLARD Directeur de Recherche, INRA, Clermont Ferrand, France Unité Mixte de Recherche 1095 INRA-Université Blaise Pascal Génétique, Diversité et Ecophysiologie des Céréales 63039 Clermont Ferrand ABSTRACT / RESUME _____________________________________________________________________________ Proteomic analysis of endosperm and peripheral layers during kernel development of wheat (Triticum aestivum L.) and a preliminary approach of data integration with transcriptome Wheat is the second most produced cereal in the world, important for food, feed and many industrial uses. Understanding of the mechanisms involved in grain development is fundamental for developing high quality wheat. In particular, detailed knowledge of the wheat grain physiology and molecular mechanisms involved in its development would help in breeding not only of wheat but also many other cereals. A proteomic approach has been used in this context but, up to now, there had been no work on developing tissues at very short temporal distances. This thesis presents, firstly, a proteomic study to characterize protein expression changes in peripheral layers and in starchy endosperm of wheat, during kernel development. We used grains of Triticum aestivum cv Récital, cultivated at INRA, Clermont-Ferrand. Grains were harvested at each 50°Cd from fertilization to maturity at fifteen stages for peripheral layers and at twenty-one stages for starchy endosperm. After grain dissection, protein extraction and 2DE- MALDI-TOF MS and data mining, we identified 207 differentially expressed proteins at fifteen stages (0°Cd-700°Cd) of peripheral layers during kernel development. These proteins were then classed in sixteen different functional classes. HCA revealed five different expression profiles during development. Similarly after obtaining starchy endosperm from dissected grains, we performed protein extraction specific to metabolic proteins. After 2DE, 487 proteins were identified from fertilization to grain maturity (0°Cd-1006°Cd), using LC-MS and data mining. Proteins were grouped in nine different expression profiles and were classed in seventeen biochemical functions. We have constructed proteome maps of these two important grain tissues during kernel development. Further, the comparison of peripheral layers and starchy endosperm proteomic data was made, with an objective to understand whether the changes in different biochemical processes differ between these tissues. Finally, we performed an integration of our proteomic data (focusing our approach on proteins involved in carbohydrate metabolism) with that of transcriptomics. Only 32% of proteome/transcriptome expression profiles showed a significant correlation during development (from 152°Cd-700°Cd). Comparison of enzyme expression profiles with those of proteome and transcriptome would help to distinguish the processes regulated at transcriptome level and those controlled at the proteome level. This comprehensive grain development data could further help in construction of a Récital databank, which may be used as reference for studies of diseased and stressed grain tissues during development. Keywords: Triticum aestivum, grain development, peripheral layers, starchy endosperm, metabolic proteins, expression profiles, proteome/transcriptome Analyse du protéome de l’albumen et des couches périphérique du grain de blé (Triticum aestivum L.) en développement: vers une intégration des données avec le transcriptome Le blé est la seconde céréale la plus produite dans le monde. Il constitue une importante source de denrées alimentaires et de beaucoup d’autres usages industriels. La compréhension des mécanismes impliqués dans le développement du grain de blé est fondamentale pour développer des blés à valeur ajoutée. La physiologie du grain de blé et les mécanismes moléculaires impliqués dans son développement nécessitent d’être mieux connus et ces connaissances pourront être très utiles pour l’amélioration du blé mais aussi des autres céréales. L’approche protéomique a été aussi utilisée dans ce contexte mais aucun travail n’avait jusqu’ici été réalisé sur la totalité des phases de développement des tissus et sur des intervalles de temps très courts. La caractérisation des changements d’expressions protéiques dans les couches périphériques du grain et de l’albumen est présentée dans cette étude. Nous avons utilisé les grains de Triticum aestivum de la variété Récital, cultivés à l’INRA de Clermont- Ferrand. Les grains ont été prélevés tous les 50°C jour (°Cj) depuis la fécondation jusqu’à la maturité sur 15 stades de développement pour les couches périphériques et sur 21 stades pour l’albumen amylacé. Pour chaque échantillon, les couches périphériques des grains ont été disséquées et les protéines totales extraites. L’analyse des protéines en électrophorèse bidimensionnelle puis par spectrométrie de masse MALDI-TOF a permis d’identifier via l’interrogation des bases de données, 207 protéines différentiellement exprimées sur 15 stades de développement (0°Cj-700°Cj). Ces protéines ont ensuite été classées en 16 classes fonctionnelles. L’analyse en cluster a révélé 5 profils d’expression au cours du temps. Parallèlement, l’albumen amylacé a été isolé des grains et les protéines métaboliques de ce tissu extraites. Après électrophorèse bidimensionnelle des protéines, 487 protéines variant significativement dans l’albumen sur l’ensemble des stades de développement (0°Cj-1006°Cj) ont été identifiées par utilisation de la LC-MS. Les protéines ont été réparties sur neuf profils d’expression et 17 fonctions biochimiques. Le protéome des couches periphériques a ensuite été comparé au protéome de l’albumen dans le but de comprendre si l’évolution des processus biochimiques diffère dans chacun de ces tissus. Au final, nous avons optimisé l’intégration des données protéomiques avec celles du transcriptome (en se focalisant sur les protéines du métabolisme carboné). Seulement 32% des profils d’expression protéome/transcriptome montrent une corrélation significative au cours du développement (152°Cj- 700°Cj). Les profils d’expression des enzymes ont été comparés sur les deux niveaux. Ils devraient permettre de distinguer les processus régulés au niveau du transcriptome de ceux régulés au niveau du protéome. L’ensemble de ces données pourra être compilé dans une base de données propre de la variété Récital et utilisé comme référence dans l’étude des maladies et des stress abiotiques des tissus du grain de blé en développement. Mots-clés : Triticum aestivum, grain en développement, couches périphériques, albumen amylacé, protéines métaboliques, profils d’expression, lien des données protéome/transcriptome. REMERCIEMENTS _____________________________________________________________________________________ ACKNOWLEDGMENTS This dissertation would not have been possible without the guidance and the help of several persons who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study. I take this opportunity to pay my humble regard to Dr. Gilles CHARMET (former director of the UMR, Génétique, Diversité et Ecophysiologie des Céréales) for introducing me to his elite group of scientists.
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