1 Année 2013 Thèse N° 2064 THESE pour le DOCTORAT DE L’UNIVERSITE BORDEAUX 2 Ecole Doctorale des Sciences de la Vie et de la Santé de l’Université Bordeaux-Segalen Spécialité: Biologie Végétale Hormonal regulation during initial berry development in grapevine par María Francisca Godoy Santin Née le 20 Février 1985 à Santiago, Chili Soutenue publiquement le 15 Novembre, à Pontificia Universidad Católica de Chile, Santiago, Chili Membres du Jury : Pr. Alexis KALERGIS, Universidad Católica de Chile Pr. Michael HANDFORD, University of Chile Rapporteur Pr. Claudia STANGE, University of Chile Rapportrice Pr. Loreto HOLUIGUE, Universidad Católica de Chile Examinatrice Pr. Serge DELROT, Université Victor Ségalen Bordeaux2 Examinateur Pr. Patricio ARCE-JONHSON, Universidad Católica de Chile Co-Directeur de thèse Dr. Virginie LAUVERGEAT, Université Bordeaux1 Co-Directrice de thèse 2 Acknowledgements I would like to thank my advisors Dr Patricio Arce and Dr Virginie Lauvergeat for their guidance, patience and help at every step of the way. I also want to specially acknowledge to all my labmates for their ideas, support, laughs and much more: Jenn, Xime, Amparo, Mindy, Anita, Felipe, Tomás, Susan, Mónica, Jessy, Claudia, Dani H, and everyone else who made the working place the best one ever. Special thanks to Consuelo, who guided me from the first day, and to Anibal, who has been an amazing help during these lasts years. I want to thank Nathalie Kühn for all her ideas, discussion, company and help during these experiments, where she played a fundamental part. Also, I am very grateful to my other lab across the sea, in INRA, for receiving me there: Mariam, María José, Julien, Eric, Pierre, Le, Huan, Messa, David, Fatma and all the staff for all their help. Also I am very grateful to Serge Delrot, who permitted this internship. Special thanks to Michell Hernould and Emeline Teyssier for all their help. I would like to thank Gonzalo, Catherine and Carlos for all their support during the grapevine flowering seasons, for without them all this would have been impossible. I want to thank all my friends who supported me through all these years: Estefanía, Roberto, Felipe, Alejandro, Esteban, Fabián, María José, Jano, Francisco, Javi, Francisca Cobo, Consuelo, Macarena, Anita, Ariel, Kelly, Naty Delherbe, Cristóbal, Sofía and Fabián Valdebenito, a very important person to me. Also, I would like to express my deepest gratitude to my family: my parents Jaime and Julia, and Cony, Jaime and Sebastian, who have been kind, understanding and very patient with me during all this years. This work would not have been possible without the financial support of the four-year Doctoral scholarship and a Doctoral thesis support scholarship of CONICYT. This work was supported by Fondecyt 110079 and Millenium Nucleus on Plant Functional Genomics P10-062-F. 3 This thesis is dedicated to my parents for all their love and support and to my brother Sebastián, who taught me never to give up. 4 5 Subject Index Acknowledgements……………………………….……………………………………………2 Subject Index…………………………………………………………………………………. 5 Figure Index………………………………………………………………………………….. 7 Table Index…………………………………………………………………………………… 9 Glossary……………………………………………………………………………………….10 General abstract (Spanish)..…………………………………………………………………..11 1. CHAPTER I: Bibliographic synthesis of events involved in early development of the grapevine berry: morphological, molecular and hormonal aspects ……….. 13 Abstract (French)……………………………………………………………………...14 Abstract (English)……………………………………………………………………..16 Introduction……………………………………………………………………...........17 Reproductive landmarks………………………………………………………………18 Morphological landmarks……………………………………………………………..24 Hormonal regulation during early berry development………………………………..28 Genetic landmarks: the beginning? ............................................................................ 31 Conclusions…………………………………………………………………………....34 References………………………………………………………………………….….35 2. CHAPTER II: Problem statement………………………………………………… 39 Problem statement: hypothesis and objectives………………………………………..40 3. CHAPTER III: Transcriptomic and hormonal regulation in early berry development: from pollination to fruitset………………………………………………………………………………. 43 Abstract (French)………………………………………………………………….… 44 Abstract (English)………………………………………………………………….….46 6 1. Introduction ………………………………………………………………….….48 2. Materials and methods…………………………………………………………..53 2.1 Flower emasculation, sampling and hormone inhibitor treatments of ovaries and fruits ……………………………………………………....…...……….…...53 2.2 Sample fixation and microscopy observation ….………...…………………53 2.3 RNA extraction, cDNA synthesis and PCR analysis………………………..54 2.4 RNAseq analysis…………………………………………………………….57 2.5 Quantitative comparison of gene expression………………………….........58 2.6 Hormone measurements…………………………………………………….58 3. Results…………………………………………………………………………..60 3.1 Morphological changes during early berry development: cell division and cell expansion………………………………………………………………………..60 3.2 Global gene expression from pollination to fruitset: a dynamic process……………………………………………………………….……….....66 3.3 Hormone-related gene expression during early berry development: the importance of auxins and gibberellins…………………………………………...85 3.4 Main hormones involved during initial berry development….………..........89 3.5 Effect of hormone inhibitors in fruitset achievement…...…………………...94 4. Discussion……………………………………………….………...…………….98 4.1 Mesocarp development due to cell division and expansion triggered by pollination………………………………………………………..……………..........98 4.2 Early berry development presents a dynamic gene expression profile……………………………………………………………………….………...99 4.3 New insights into hormone regulation during initial grape berry development………………………………………………………………………….101 4.4 Fruitset establishment in grapevine ….………..………………………..…...107 5. Conclusions………………………………………………………………………..111 6. References ..………………………………………………………………….……112 7. Supplemental Material…………….………………………………………………121 7 Figure index Chapter I Figure 1: Reproductive traits and events occurring shortly after pollination at anthesis………………………………………………………………………………...20 Figure 2: Expression of a fertilization marker during initial berry development…….23 Figure 3: Definition of fruitset by changes in berry volume during inittial berry development…………………………………………………………………………...26 Figure 4: Grapevine inmature berry seen by light microscopy ………………………27 Figure 5: Main features defining early berry development in grapevine……………..30 Figure 6: Transcript abundance changes of putative cell cycle related genes during initial grapevine berry development…………………………………………………...33 Chapter III Figure 1: Emasculation and hand-pollination technique on Vitis vinifera cv. Red Globe flowers………………………………………………………………………………..56 Figure 2: Effect of pollination/fertilization on fruit and mesocarp development in grapevine……………………………………………………………………………..61 Figure 3: Changes in mesocarp morphology triggered by pollination/fertilization in grapevine……………………………………………………………………………..64 Figure 4: Gene Ontology distribution of differentially expressed genes found during sequential time-points of initial berry development……………………………….…68 Figure 5: Venn diagrams of differentially expressed transcripts during early berry development in grapevine……………………………………………………………70 Figure 6: MIPS categories distribution for differentially expressed genes during early berry development…………………………………………………………………...73 Figure 7: Cluster of differentially expressed genes through early berry development………………………………………………………………………….74 Figure 8: Over-representation of selected categories during early stages of berry development………………………………………………………………………….79 8 Figure 9: Over-representation of hormone-specific categories during early berry development in grapevine……………………………………………………………..81 Figure 10: Hierarchical cluster of auxin and gibberellin-related genes during early berry development ..…………………………………………………………………...83 Figure 11: Expression of hormone related genes during early steps of berry development…………………………………………………………………………...77 Figure 12: Auxin and auxin-related compounds during early grapevine berry development………………………………………..………………………………….91 Figure 13: Gibberellins, abscisic acid and trans-Zeatin levels at initial stages of berry development………………………………………………………………………...…92 Figure 14: Levels of bioactive hormones measured from anthesis to fruitset in grapevine….…………………………………………………………………………...93 Figure 15: Inhibition of gibberellin biosynthesis (with Paclobutrazol) and auxin induced responses (with IAA-Trp) and its effect in fruitset……………………….….96 Figure 16: Fruitset establishment model in early grapevine berry development……………………………………………………………………...…..110 9 Supplemental Material Supplemental Figure 1: Cluster of differentially expressed genes through early berry development ………………………………………………....……………………………..121 Supplemental Table I: Primers used for gene amplification....……………………………..123 Supplemental Table II: Genes with differential expression during early berry development in grapevine…………………………………………………………………………………….124 10 Glossary 2-4D, Dichlorophenoxi-Acetic Acid 4-CPA, 4-Chlorophenoxy-Acetic Acid ABA, Abscisic Acid ASB1, Anthranilate Synthase Subunit 1 BRs, Brassinosteroids CKs, Cytokinins CS, Castasterone DAE, Days After Emasculation DAF, Days After Flowering DAP: Days After Pollination DPA, Days Post Anthesis DPP, Days Post Pollination ENP, Emasculated Not Pollinated GA3, Gibberellin A3 GAs, Gibberellins IAA, Indole-3-Acetic Acid PAC: Paclobutrazol PCR: Polymerase