Title Page
Contents
ABSTRACT 9
Chapter 1. HOS15 negatively regulates DIL9 to modulate ABA-dependent drought tolerance in Arabidopsis 11
1. Introduction 11
1.1. Plant response to stress. 11
1.2. Plant response to drought stress. 13
1.3. ABA accumulation in drought stress. 15
1.4. ABA biosynthesis role in drought stress. 16
2. Material and Methods 22
2.1. Plant materials and growth conditions 22
2.2. Seedling's Growth Experiments 22
2.3. Co-Immunoprecipitation (Co-IP) assay 23
2.4. Luciferase Assays 23
2.5. Generation of DIL9-CRISPR/Cas9 mutants' lines. 24
2.6. Drought and Water/Weight loss assays 25
2.7. ChIP Experiments 25
2.8. qRT and RT-PCRs Analysis 25
2.9. Yeast Two-Hybrid Assay 26
2.10. Stomata Aperture Assay 26
2.11. Statistical Analysis 27
2.12. Accession numbers 27
3. Results 30
3.1. DIL9 is an interacting partner of HOS15. 30
3.2. HOS15 regulates ABA-induced DIL9 proteasomal degradation. 32
3.3. ABA stabilized the interaction between DIL9 and HOS15. 35
3.4. Drought reduces DIL9 proteasomal degradation. 36
3.5. Generation and validation of DIL9-Cas9 mutants' lines. 38
3.6. DIL9 is epistatic to HOS15 in drought and ABA traits. 39
3.7. DIL9 direct associate with the promoter and activates expression of NCED3. 43
3.8. DIL9 regulates drought and ABA responses independent of OST1. 46
4. Discussion and Conclusion 48
5. Working model 52
Chapter 2. HOS15 modulates DIL9 activity to regulate SA-mediated drought-tolerance in Arabidopsis 53
ABSTRACT 53
1. Introduction 55
2. Materials and Methods 58
2.1. Plant materials and growth conditions 58
2.2. Seedling's treatment with ABA 58
2.3. RNA isolation and cDNA synthesis 58
2.4. Drought and water/weight loss assays 59
2.5. ChIP assay 59
2.6. Gene expression analysis 60
2.7. Stomata aperture assay 60
2.8. RNA-Seq data analysis 61
2.9. Statistical analysis 61
2.10. Accession numbers 61
3. Results 62
3.1. Drought stress encourages plants defense-responsive genes. 62
3.2. Drought stress elevated PR genes in hos15-2 plants. 64
3.3. HOS15 impairs DIL9 activity to the PR genes transcripts. 66
3.4. HOS15 interferes with DIL9 to associate with PR genes promoter. 68
3.5. ABA-reduced DIL9 activity to the PR genes. 70
4. Discussion and Conclusion 72
5. Working model 75
References 76
Appendix 93
ABSTRACT (in Korean) 94
Table 1. List of Primers 28
Table 2. List of Appendix 93
Figure 1. Plant adaptive responses to abiotic stress. 12
Figure 2. Representative chart for drought stress effect on plants. 14
Figure 3. Schematic representation of ABA biosynthesis pathway. 17
Figure 4. HOS15 is a multifunctional protein. 20
Figure 5. DIL9 interaction with HOS15 protein. 31
Figure 6. Western Blot (WB) assay for the DIL9 protein stability. 34
Figure 7. DIL9 and HOS15 interaction in ABA. 35
Figure 8. Western Blot (WB) assays for HOS15 and DIL9 abundance in drought stress. 37
Figure 9. Generation and validation of DIL9-CRISPR/Cas9 mutants 38
Figure 10. Genetic relation between DIL9 and HOS15 in drought and ABA. 40
Figure 11. DIL9-GFP/hos15 enhances the plant's drought stress response and ABA traits. 42
Figure 12. DIL9 regulates NCED3 in drought stress. 45
Figure 13. DIL9 regulates drought and ABA independent of OST1. 47
Figure 14. Working model for HOS15-DIL9-NCED3 role in drought. 52
Figure 15. Transcriptomic analysis for drought-treated plants. 63
Figure 16. PR genes expression in drought stress. 65
Figure 17. PR genes transcripts in DIL9-GFP and DIL9-GFP/hos15-2 plants in a drought. 67
Figure 18. HOS15 interferes with DIL9 for the PR genes. 69
Figure 19. DIL9 activity to the PR genes in ABA. 71
Figure 20. Proposed model for DIL9 and HOS15 function in drought stress. 75