In planta maternal haploid induction by MTL/NLD/ZmPLA1 homologous gene editing and enhancement of turnip crinkle virus resistance by overexpressing pPLAIIIα = MTL/NLD/ZmPLA1 상동유전자 교정에 의한 모계 유래 반수체 유도 및 pPLAIIIα 과발현에 의한 순무주름바이러스 저항성 증가에 관한 연구

목차 (Table of Contents)

• Table of contents i
• List of figures and tables v
• Abbreviations ix
• Abstract 1
• Chapter 1. General introduction 4
• 1. Patatin-related phspholipase A and its roles in plants 4
• 1.1. Patatin-related phospholipase A in haploid induction 11
• 1.1.1. Doubled haploid technology 11
• 1.1.2. Phospholipase-mediated haploid induction 18
• 1.1.3. Types of haploid inducer and hypotheses about their underlying mechanisms 20
• 1.2. Patatin-related phospholipase A in regulating cell wall composition 28
• 1.3. Patatin-related phospholipase A in plant defense 31
• 2. References 35
• Chapter 2. Loss-of-function of gynoecium-expressed phospholipase pPLAIIγ triggers maternal haploid induction in Arabidopsis 47
• Abstract 47
• 1. Introduction 48
• 2. Materials and methods 52
• 2.1. Plant materials and growth conditions 52
• 2.2. Transgenic constructions and in planta transformation 52
• 2.3. β-glucuronidase (GUS) histochemical analysis 53
• 2.4. Observation of reporter gene expression 54
• 2.5. Total RNA extraction and quantitative real-time PCR (qPCR) 54
• 2.6. Ploidy analysis using flow cytometry 55
• 2.7. Karyotyping 56
• 2.8. RNA-seq profiling and analysis 56
• 2.9. DAPI staining for visualization of ovule nuclei 57
• 3. Results 58
• 3.1. Loss-of-function of pPLAIIγ triggers in planta haploid induction in dicot Arabidopsis 58
• 3.2. A female in vivo haploid induction system by pPLAIIγ mutation triggers maternal haploid plants 71
• 3.3. Gynoecium-expressed pPLAIIγ localized in plasma membrane connected with Hechtian strands 76
• 3.4. PIN1 and PIN3 are expressed in the funiculus and their PM localization is altered in sgpPLAIIγ 81
• 4. Disscusion 87
• 4.1. Comparison with other in vivo haploid inducer systems MTL/NLD/ZmPLA1, DMP, and CENH3 88
• 4.2. Altered localization of PIN auxin transporter in the funiculus of pPLAIIγ mutation caused haploid induction 91
• 5. References 101
• Chapter 3. Loss-of-function of pollen-expressed OsMATL2 triggers in planta maternal haploid in japonica rice 110
• Abstract 110
• 1. Introduction 111
• 2. Materials and methods 112
• 2.1. Plant materials and growth conditions 112
• 2.2. Transgenic construction and in planta transformation 112
• 2.3. β-glucuronidase (GUS) histochemical analysis 113
• 2.4. Observation of reporter gene expression 113
• 2.5. Ploidy analysis using flow cytometry 113
• 2.6. Chromosome spread analysis 114
• 3. Results and discussion 115
• 3.1. OsMATL2 is abunduntly expressed in pollen 116
• 3.2. Construction of CRISPR-Cas9-mediated OsMATL2 edited lines 122
• 3.3. sgOsMATL2 lines induce haploid progeny by self-pollination 124
• 3.4. Intraspecific cross reveals that sgOsMATL2 induces maternal haploid 126
• 4. Conclusion 131
• 5. References 133
• Chapter 4. Phospholipase pPLAIIIα increases germination rate and resistance to turnip crinkle virus when overexpressed 135
• Abstract 135
• 1. Introduction 137
• 2. Materials and methods 140
• 2.1. Plant materials and growth conditions 140
• 2.2. Transgene constructs and Arabidopsis transformation 140
• 2.3. GUS histochemical analysis 141
• 2.4. Observation of reporter gene expression 141
• 2.5. Gene expression analysis by quantitative real time PCR (qPCR) 141
• 2.6. Lipid extraction and lipidomic analysis 142
• 2.7. Protein purification/extraction, and immunoblotting 144
• 2.8. In vitro enzymatic assays 145
• 2.9. 3,3'-Diaminobenzidine (DAB) staining 145
• 2.10. Turnip crinkle virus infection and HR response analysis 146
• 2.11. Phytohormone analysis 146
• 3. Results 147
• 3.1. Despite lacking canonical motifs, pPLAIIIα has retained lipase activity 147
• 3.2. Spatial and temporal expression patterns of pPLAIIIα 149
• 3.3. Knockout, overexpression and subcellular localization of Arabidopsis pPLAIIIα 151
• 3.4. Lipidomics reveal a wide range of acyl-lipids are decreased in pPLAIIIαOE on a seedling weight basis 157
• 3.5. pPLAIIIα expression level affects the size and shape of many cells and organs 164
• 3.6. pPLAIIIαOE is involved in the control of seed size and germination 170
• 3.7. Transcript levels of ethylene and GA biosynthesis genes increased during pPLAIIIαOE seed i mbibition 173
• 3.8. Overexpression of pPLAIIIα delayed senescence by reducing ROS 175
• 3.9. Overexpression of pPLAIIIα confers turnip crinkle virus resistance by altering salicylic acid (SA) and jasmonic acid (JA) contents 177
• 4. Discussion 181
• 4.1. Lipid substrates of pPLAIIIα in vivo 182
• 4.2. PA levels and PLD expression 185
• 4.3. Role of pPLAIIIα in cell elongation and plant growth 188
• 4.4. Role of pPLAIIIα in seed germination 190
• 4.5. Reduced PA and JA levels may explain the low level of ROS in pPLAIIIαOE 191
• 4.6. A higher SA to JA ratio may increase the resistance of pPLAIIIαOE to turnip crinkle virus 192
• 5. References 194
• Abstract (in Korean) 200
• Acknowledgements 202
• Curriculum vitae 203