CONTENTS
SUMMARY ... 1
RÉSUMÉ ... 2
LIST OF ABBREVIATIONS ... 3
LIST OF FIGURES ... 5
LIST OF TABLES ... 7
CHAPTER ONE : GENERAL INTRODUCTION ... 8
1.1. Context of the work ... 8
1.2. Diversity of RING-domains ... 13
1.3. Ubiquitination ... 20
1.3.1. Ubiquitination pathway ... 21
1.3.2. Ubiquitination proteins in plants ... 22
1.3.2.1. Ubiquitin ... 22
1.3.2.2. Ubiquitin activating enzymes (E1) ... 26
1.3.2.3. Ubiquitin conjugating enzymes (E2). ... 27
1.3.2.4. Ubiquitin ligases (E3) ... 29
1.3.3. Types of ubiquitination ... 34
1.3.3.1. Monoubiquitination ... 35
1.3.3.2. Polyubiquitination ... 39
1.4. Objectives of the work ... 41
CHAPTER TWO: IN VITRO UBIQUITINATION ASSAYS INDICATE THAT RECOMBINANT POPLAR PTARHE1 IS A FUNCTIONAL E3 LIGASE ... 42
2.1. Introduction ... 42
2.2. Materials and methods ... 43
2.2.1. RNA extraction and cDNA synthesis ... 43
2.2.2. Production of PtaRHE1 and PtaRHE1-Ct recombinant proteins ... 44
2.2.3. In vitro auto-ubiquitination assay ... 45
2.2.4. Cloning of poplar ubiquitin conjugating enzyme UbC5a. ... 46
2.3. Results ... 47
2.3.1. Production of PtaRHE1 recombinant proteins ... 47
2.3.2. In vitro ubiquitination assay ... 50
2.3.3. Cloning of PtaUbC5a ... 53
2.3.4. Production of recombinant PtaUbC5a ... 56
2.4. DISCUSSION ... 57
CHAPTER THREE: STUDY OF THE SPATIO-TEMPORAL EXPRESSION OF PTARHE1 AND ITS REGULATION BY VARIOUS STRESS TREATMENTS AND PTARHE1 PROTEIN INTRACELLULAR LOCALIZATION ... 60
3.1. Introduction ... 60
3.2. Materials and methods ... 61
3.2.1. PtaRHE1 promoter analysis ... 61
3.2.2. Construction of chimeric PtaRHE1 Promoter-β-Glucuronidase Gene ... 61
3.2.3. Plant material and growth conditions ... 61
3.2.4. Histochemical GUS assays... 62
3.2.5. Treatment of transgenic tobacco ... 63
3.2.6. Localization of expression of PtaRHE1 in poplar stem by in situ hybridization ... 63
3.2.6.1. Plant material ... 65
3.2.6.2. Tissue fixation and embedding in paraffin ... 65
3.2.6.3. Preparation of tissue slices ... 66
3.2.6.4. Synthesis of DIG-labeled RNA probes ... 66
3.2.6.5. Pre-hybridization ... 67
3.2.6.6. In situ hybridization with DIG labeled probes ... 68
3.2.7. Subcellular localization of PtaRHE1 in tobacco leaves epidermal cells ... 71
3.2.7.1. In silico prediction and localization of PtaRHE1 ... 71
3.2.7.2. In vivo localization of PtaRHE1 by GFP-reporting and agroinfiltration ... 71
3.2.7.2.1. Construction of PtaRHE1-GFP reporter plasmid and agroinfiltration in tobacco leaves. ... 71
3.2.7.2. 2. Microscopic observations ... 72
3.3. Results ... 73
3.3.1. PtaRHE1 promoter analysis ... 73
3.3.2. pPtaRHE1::GFP::GUS plant regeneration... 77
3.3.3. Induction of PtaRHE1 promoter by various biotic and abiotic stresses ... 77
3.3.4. Expression analysis of PtaRHE1 promoter during plant development ... 80
3.3.5. Localization of PtaRHE1 by in situ hybridization in poplar stem. ... 80
3.3.6. Intracellular localization of PtaRHE1 protein ... 83
3.4. Discussion... 86
CHAPTER FOUR: OVEREXPRESSION OF PTARHE1 IN TRANSGENIC TOBACCO TRIGGERS LEAF CURLING, LEAF BLADE NECROSIS AS WELL AS GROWTH
RETARDATION AND FLOWER TRANSITION DELAY ... 89
4.1. Introduction ... 89
4.2. Materials and methods ... 89
4.2.1. Vector construction for PtaRHE1 overexpression and plant transformation ... 89
4.2.2. Total RNA extraction and cDNA obtention ... 90
4.2.3. Quantitative RT-qPCR ... 90
4.3. Results ... 95
4.3.1. Phenotype analysis ... 95
4.3.2. Drought tolerance ... 97
4.3.3. Expression analysis of defence-related proteins ... 99
4.4. Discussion... 104
5. GENERAL CONCLUSION AND PERSPECTIVES ... 107
5.1. PtaRHE1 is a functional E3 ligase ... 107
5.2. PtaRHE1 promoter-driven GUS activity is induced by various biotic and abiotic treatments and is developmentally regulated ... 109
5.3. PtaRHE1 localizes in the plasma membrane and in the nucleus ... 110
5.4. The overexpression of PtaRHE1 induces pleiotropic effects on plant growth and development ... 112
5.5. Several classes of genes encoding inducible defence related proteins are up-regulated in transgenic plants overexpressing PtaRHE1 ... 114
