Title Page
Contents
ABSTRACT 10
Literature Review 12
Chapter 1. HOS15 Regulates HLS1 Transcript Level by Mediating EIN3 Protein Degradation 22
Ⅰ. Introduction 22
Ⅱ. Material and Methods 28
2.1. Plant materials and growth conditions 28
2.2. Generation of transgenic Arabidopsis lines 28
2.3. RNA Extraction and Real-Time PCR Total 29
2.4. Yeast Two-hybrid Assay 29
2.5. Immunoblot analysis 29
2.6. Immunoprecipitation assay 30
2.7. Luciferase Complementation Imaging (LCI) Assay 30
2.8. Measurement of apical hook angle curvature 31
2.9. ChIP Assay 31
Ⅲ. Results 35
3.1. hos15-2 mutant shows severe hook formation 35
3.2. hos15-2 doesn't alter ET signaling pathway genes expression 37
3.3. HOS15 interacts with EIN3 39
3.4. HOS15 negatively regulates EIN3 protein stability 44
3.5. Expression pattern of hook formation gene 46
3.6. HOS15 negatively regulates HLS1 expression 48
3.7. ein3-1, eil1-1 mutation suppressed the hos15-2 severe apical hook formation 50
Ⅳ. Discussion and Conclusion 52
Ⅴ. Working model for HOS15 in Ethylene signaling 57
Chapter 2. HOS15 Fine-Tunes EIN3 protein Stability for Positive Regulation of Cold Signaling 59
Ⅰ. Introduction 59
Ⅱ. Materials and Methods 65
2.1. Immunoprecipitation assay 65
2.2. Immunoblot assay 65
2.3. RNA Extraction and Real-Time PCR Total 65
2.4. ChIP Assay. 66
2.5. RNA-Seq and ChIP-Seq assay 66
2.6. Freezing Tolerance Assay 66
Ⅲ. Results 68
3.1. Expression of EIN3 and EIL1 under cold stress 68
3.2. HOS15 positively regulates the expression of ARRs 69
3.3. Cold mediates EIN3 interaction with HOS15 70
3.4. Under cold stress EIN3 degradation is mediated by HOS15 71
3.5. EIN3 binding to promoters of ARRs genes is elevated in hos15-2 73
3.6. Cold stress induces miP1a and miP1b transcript level 75
3.7. ARRs genes expression in miP1a miP1b double mutant 77
3.8. miP1a miP1b double mutant lines display the cold-sensitive phenotype 79
Ⅳ. Discussion and Conclusion 81
Ⅴ. A Proposed Working Model for EIN3 regulation in cold stress 85
References 87
Abbreviations 101
ABSTRACT (in Korean) 103
Table 1. List of Primers 32
Figure 1. Plant exposure to various Abiotic and Biotic stresses. 14
Figure 2. The transmitting of ET signaling pathway in Arabidopsis. 16
Figure 3. ET biosynthesis pathway in plants. 18
Figure 4. Plant ET responses under abiotic, biotic stresses and during developmental changes. 21
Figure 5. Model for ethylene signaling and EIN3 protein accumulation. 23
Figure 6. A Working model for the regulation of EIN2-C in ET presence. 24
Figure 7. HOS15 serves multiple functions protein and has a diverse range of roles. 26
Figure 8. hos15-2 plants are highly sensitive to ET. 36
Figure 9. Expression of genes in the ET signaling pathway. 38
Figure 10. HOS15 does not interact with EIN3. 40
Figure 11. EIN3 (P) physically interacts with HOS15. 42
Figure 12. HOS15 interacts with EIN3. 43
Figure 13. HOS15 fine-tune the EIN3 protein stability. 45
Figure 14. HLS1 gene expression in hos15-2. 47
Figure 15. EIN3 binding to HLS1 promotor. 49
Figure 16. ein3-1 is epistatic to hos15-2. 51
Figure 17. A Proposed working model. 57
Figure 18. The CBFs dependent and independent cold signaling pathways. 61
Figure 19. EIN3-mediates a network of signals. 63
Figure 20. EIN3, and EIl1 transcript expression is not regulated by HOS15 during cold stress. 68
Figure 21. ARR5, ARR7, and ARR15 transcript expression is indirectly regulated by HOS15 during cold stress. 69
Figure 22. Interaction between EIN3 interacts with and HOS15. 70
Figure 23. Cold induces degradation of EIN3 is impaired in hos15-2. 72
Figure 24. EIN3 association to ARRs promoter. 74
Figure 25. Genes transcript expression of miP1a/b during cold. 76
Figure 26. Genes expression of ARRs during cold in miP1a miP1b double mutant. 78
Figure 27. miP1a miP1b are sensitive to cold stress. 80
Figure 28. A working model for EIN3 regulation in cold stress. 86