목차

Abstract 1
Chapter 1 3
Introduction 3
1.1. Production of redox signalling molecules in plants 3
1.2. Homeostasis of redox signalling molecules in plants 6
1.3. ROS; Key players during plant-pathogen interaction 8
1.4. ROS; Key players during plant-symbiont interaction 10
1.5. The era of nitric oxide (NO) 12
1.6. Production of NO in animals and plants 13
1.7. Challenges in the identification of nitric oxide synthase in plants 17
1.8. NO; A key player of RNS during plant-pathogen interaction 17
1.9. NO; A key player of RNS during plant-symbiont interactions 19
Chapter 2 21
Generalized materials and methods 21
2.1. Plant materials 21
2.2. Surface sterilization of the seeds 22
2.3. Soil preparation and plant growth 22
2.4. Preparation of MS (Murashige and Skoog) medium 23
2.5. Preparation of redox-stress media (for redox stress assay) 24
2.5.1. Preparation of methyl viologen (MV)-supplemented ½ MS media 24
2.5.2. Preparation of hydrogen peroxide (H2O2)-supplemented ½ MS media 25
2.5.3. Preparation of S-nitroso L-Cysteine (CySNO)-supplemented ½ MS media 25
2.5.4 Preparation of GSNO-supplemented media 26
2.6. Seeds sowing and plant growth conditions (Redox stress assay) 27
2.7. Preparation of Luria Bertani (LB) media 28
2.8. Genotyping for the confirmation of homozygous lines 29
2.8.1. Designing of primers for the confirmation of T-DNA insertion and homozygosity of the mutant lines 30
2.8.2. Preparation of 2X CTAB buffer 31
2.8.3. Protocol for the extraction of genomic DNA using CTAB 32
2.8.4. Preparation of 1% agarose gel 33
2.8.5. Gel images assessment in gel documentation unit 33
2.9. Extraction of RNA by TRIzol reagent method (Life technologies) 34
2.10. Synthesis of cDNA using DiaStar RT Kit (Solgent) 35
2.11. Growth of the pathogens, inoculation and electrolyte leakage assay 36
2.12. Preparation of Escherichia coli competent cells 38
2.13. In-silico analysis of the studied genes 39
Chapter 3 40
The Role of Nitric Oxide-induced AtILL6 in Growth and Disease Resistance in Arabidopsis thaliana 40
3.1. Abstract 40
3.2. Introduction 41
3.3. Materials and methods 44
3.3.1. Plant materials and growth conditions 44
3.3.2. Oxidative and nitro-oxidative stress conditions 45
3.3.3. Pathogen growth and inoculation and electrolyte leakage assay 45
3.3.4. Quantitative real-time PCR (qRT-PCR) analysis 46
3.3.5. Statistical analysis 47
3.4. Results 48
3.4.1. Confirmation of the T-DNA insertion in the mutant line atill6 48
3.4.2. Verification of the abolishment of the AtILL6 gene expression in the atill6 mutant line 49
3.4.3. AtILL6 differentially regulates root and shoot length under oxidative and nitro-oxidative stress conditions 49
3.4.4. AtILL6 positively regulates plant basal defense 51
3.4.5. AtILL6 positively regulates R-gene-mediated resistance 53
3.4.6. AtILL6 positively regulates SAR 54
3.5. Discussion 57
3.6. Conclusion 59
Chapter 4 60
The Role of Nitric Oxide-Induced AtBSMt1 in Growth and Systemic Acquired Resistance of Arabidopsis thaliana 60
4.1. Abstract 60
4.2. Introduction 62
4.3. Materials and Methods 64
4.3.1. Plant materials and growth conditions 64
4.3.2. Oxidative and nitro-oxidative stress experiments 65
4.3.3. Growth of the pathogens, inoculation and electrolyte leakage assay 66
4.3.4. SAR (Biological experiment) 67
4.3.5. Quantitative real-time PCR (qRT-PCR) analysis 67
4.3.6. Statistical analysis 69
4.4. Results 69
4.4.1. Confirmation of the T-DNA insertion in the atbsmt1 mutant line 69
4.4.2. AtBSMT1 differentially regulates CDF, root, and shoot length under control, oxidative, and nitro-oxidative stress conditions 70
4.4.2. AtBSMT1 has no contribution to basal defense 72
4.4.3. AtBSMT1 has no contribution to R-gene-mediated resistance 73
4.4.4. AtBSMT1 has a significant contribution to SAR 75
4.5. Discussion 77
4.6. Conclusion 79
Chapter 5 80
The role of nitric oxide in the regulation of the Shikimate pathway in plants 80
5.1. Abstract 80
5.2. Introduction 81
5.3. Materials and Methods 85
5.3.1. Plant materials and growth conditions 85
5.3.2. Oxidative and nitro-oxidative stress conditions 86
5.3.3. Growth of the pathogens, inoculation and electrolyte leakage assay 87
5.3.4. Total RNA extraction, cDNA synthesis, RT-PCR, and quantitative real-time PCR (qRT-PCR) analysis 88
5.3.5. Statistical analysis 90
5.3.6. Generation of overexpress line (OX) for AtCHS 91
5.3.7. Amplification of the AtCHS gene using Taq polymerase 91
5.3.8. PCR purification and TA-cloning 92
5.3.9. Confirmation of the insert through colony PCR and sequencing 93
5.3.10. LR-reaction 94
5.3.11. Confirmation of the insert in the destination vector (pEarleyGate 103) 95
5.3.12. Plasmid extraction (miniprep) using Qiagen Miniprep kit 95
5.3.13. Agrobacterium transformation 96
5.3.14. Floral dip process and BASTA selection 97
5.3.15. Genotyping of the knock out (KO) lines and transformed plants 98
5.4. Results 98
5.4.1. Cloning of AtCHS in pCR™8/GW/TOPO® 98
5.4.2. Cloning of AtCHS into the destination vector pEarleyGate 103 105
5.4.3. Transformation of AtCHS in Agrobacterium 111
5.4.4. The floral dip process 112
5.4.5. Confirmation of the T-DNA insertion in the atdahp and atchs mutant lines 113
5.4.6. AtDAHP and AtCHS differentially regulates CDF, root, and shoot length under control, oxidative, and nitro-oxidative stress conditions 114
5.4.7. AtDAHP and AtCHS positively regulates plant basal defense 116
5.4.8. AtDAHP and AtCHS positively regulates R-gene-mediated resistance 118
5.4.9. AtDAHP and AtCHS positively regulates SAR 120
5.5. Discussion 123
5.6. Conclusion 125
5.7. Combined bioinformatics analysis of the candidate genes 125
5.7.1. Gene descriptions of the candidate genes 126
5.7.2. Gene ontology (GO) analysis of the candidate genes 127
5.7.3. Identification of CIS regulatory elements 129
5.7.4. Network analysis to identify other interacting proteins 130
6. References 131
Abstract in Korean 154