Title Page
Contents
ABSTRACT 9
Chapter 1. Introduction 11
1.1. Plant Response to environmental Challenges 11
1.2. miRNA processing machinery under plant environmental challenges 15
1.3. Chromatin remodeling in plant environmental challenges 19
1.4. Study objectives 20
Chapter 2. Materials and Methods 22
2.1. Plant Materials and Growth Conditions 22
2.2. Yeast Two-Hybrid Assay 23
2.3. Co-Immunoprecipitation Assays (co-IP) 23
2.4. Chromatin Immunoprecipitation Assay (ChIP) 23
2.5. RNA and miRNA analysis 24
2.6. Small RNA blot analysis 27
2.7. In vitro pri-miRNA processing assay 28
2.8. Subcellular fractionation 29
2.9. Confocal microscopy 30
2.10. Split luciferase assay 31
2.11. Accession Numbers 31
Chapter 3. Results 32
3.1. A forward genetic screening identified HOS15 to be involved in miRNA biogenesis 32
3.2. HOS15 mutants displays dysregulation in the miRNA/miRNA targets 37
3.3. HOS15 exclusively interacts with HYL1 40
3.4. HYL1 interacts with HDA9 and HDA19 proteins 45
3.5. HDA9 epigenetically regulates ABA responsive MIRNA genes 48
3.6. HOS15-HDA9-HYL1 co-associate and epigenetically regulates MIRNA loci under ABA 51
3.7. HOS15 and HDA9 suppress ABA-responsive miRNA accumulation 54
3.8. HYL1 recruitment of HOS15-HDA9 complex to MIRNA relies on nascent pri-miRNA binding 59
3.9. HOS15 and HDA9-deficiency causes a negligible alteration of the levels of pri-miRNAs 64
3.10. Accelerated miRNA processing in hos15-2 and hda9-1 mutants under ABA 70
Chapter 4. Discussion 76
Chapter 5. Conclusion 82
References 84
Appendices 92
[Appendix 1] HOS15 mutants miRNAtome 92
[Appendix 2] Col-0 miRNA expression with timepoint ABA treatment. 92
[Appendix 3] Col-0, hos15, hda9, hyl1 small RNA sequencing analysis 92
[Appendix 4] hos15, hda9, hyl1 pri-miRNA sequencing analysis 92
[Appendix 5] hos15, hda9, hyl1 small RNA sequencing, pri-miRNA correlations 92
Abstract (in Korean) 93
Table 2-1. List of primers used for cloning 22
Table 2-2. List of primers used for qRT-PCR 25
Table 3-1. List of miRNAs used in Figure 3-9-1B 69
Figure 1-1. Plant Response to environmental Challenges 15
Figure 1-2. microRNA regulation of plant environmental challenges 16
Figure 1-3. microRNA expression and processing 17
Figure 3-1. Characterization of the hos15 mutant 36
Figure 3-2. HOS15 mutants displays dysregulation in the miRNA/miRNA targets 39
Figure 3-3-1. HOS15 exclusively interacts with HYL1 43
Figure 3-3-2. HYL1 accumulation in HOS15 and HDA9 mutants 44
Figure 3-4. HYL1 interacts with HDA9 and HDA19 proteins 47
Figure 3-5. HDA9 epigenetically regulates ABA responsive MIRNA genes 50
Figure 3-6. HOS15-HDA9-HYL1 co-associate and epigenetically regulates MIRNA loci under ABA 53
Figure 3-7. HOS15 and HDA9 suppress ABA-responsive miRNA accumulation 58
Figure 3-8. HYL1 recruitment of HOS15-HDA9 complex to MIRNA relies on nascent pri-miRNA binding 62
Figure 3-9-1. HOS15 and HDA9-deficiency causes a negligible alteration of the levels of pri-miRNAs 68
Figure 3-9-2. Enhanced RNA POLII association to MIRNA loci in hos15-2 and hos15-3 mutants 69
Figure 3-10-1. Accelerated miRNA processing in hos15-2 and hda9-1 mutants under ABA 73
Figure 3-10-2. Unaltered miRNA processing precision in hos15-2 and hda9-1 mutants 75
Figure 4-1. HYL1 recruits the HOS15-HDA9 chromatin modifier complex to the MIRNA genes to regulate the loci expression 80