Title Page
Contents
CHAPTER Ⅰ. LITERATURE REVIEW 13
1. BR in the plant growth and development 13
1.1. Physiological effects of BR on Plant growth and development 13
1.2. BR signaling pathway 15
2. ABA in plant growth and development 20
2.1. ABA signaling pathway 20
2.2. BAK1's role in ABA-induced stomatal closure 23
3. Proposed research 24
CHAPTER Ⅱ. Reciprocal inhibition of expression between RAV1 and BES1 modulates plant growth and development in Arabidopsis 27
1. Abstract 27
2. Introduction 28
3. Materials and methods 32
3.1. Plant materials and growth conditions 32
3.2. Generation of transgenic plants 32
3.3. Quantitative RT-PCR and gene expression analyses 33
3.4. EMSA assay 33
3.5. Luciferase assay 34
3.6. ChIP-qPCR assay 35
3.7. Western blot analysis 35
3.8. RNA-seq analysis 36
3.9. Statistical analysis 37
4. Results 43
4.1. RAV1 overexpression reduced the BR signaling capacity of Arabidopsis plants 43
4.2. BES1 directly binds to RAV1 promoter regions, resulting in the repression of RAV1 expression 49
4.3. RAV1 binds directly to the BES1 promoter region, resulting in repression of BES1 expression 55
4.4. BR-constitutive phenotypes in bes1-D mutant was reduced by the overexpression of RAV1 63
4.5. Transcriptome analyses revealed that RAV1 and BES1 oppositely regulate some target gene sets 70
5. Discussion 79
CHAPTER Ⅲ. Brassinosteroid-insensitive 1-associated receptor kinase 1 modulates abscisic acid signaling by inducing PYR1 monomerization and association with ABI1 in Arabidopsis 87
1. Abstract 87
2. Introduction 88
3. Materials and methods 92
3.1. Preparation of Plant materials 92
3.2. Plasmid Construction 93
3.3. Transient Expression in Tobacco Leaves 94
3.4. In vitro Kinase Assay 95
3.5. Luciferase Reporter Assay 95
3.6. [³H]-Abscisic Acid-Binding Assay 96
3.7. Co-immunoprecipitation and Western Blot Analysis 97
3.8. Pull-Down Assay 97
3.9. Yeast Two-Hybrid Assay 98
3.10. Stomatal Aperture Measurements 98
3.11. Determination of Seed Germination 99
3.12. Measurement of Root Growth 99
3.13. Drought Tolerance Assessment 99
3.14. Quantitative RT-PCR 100
4. Results 105
4.1. BAK1 interacts with PYR1 in vitro and in vivo 105
4.2. BAK1 Phosphorylates PYR1 on T137 and S142 Residues 110
4.3. PYR1DD and PYR1AA Variants Affect Abscisic Acid Responses in Plants 120
4.4. Phosphomimetic PYR1 Showed Increased Abscisic Acid Binding Capacity 130
4.5. PYR1 Phosphorylation by BAK1 Induced Increased PYR1 Dimer Dissociation 134
4.6. BAK1 Phosphorylation on PYR1 Increases PYR1 and ABI1 Interactions 139
5. Discussion 147
CHAPTER Ⅳ. Summary and Conclusion 155
REFERENCES 160
ABSTRACT IN KOREAN 176
Table 1. Primer sequences used in chapter II 38
Table 2. Primer sequences used in chapter III 101
Figure 1. Overview of BR signaling pathway 18
Figure 2. Overview of ABA signaling pathway 22
Figure 3. BL sensitivity was monitored in BR-biosynthetic or BR-signaling mutants 45
Figure 4. The overexpression of RAV1 resulted in the reduction of BL sensitivity in plants 47
Figure 5. BES1 binds to the RAV1 promoter and represses its expression 52
Figure 6. The EMSA assay shows that BES1 binds to several sites in the RAV1 promoter 54
Figure 7. RAV1 binds to the BES1 promoter and represses its expression 58
Figure 8. The EMSA assay shows that RAV1 binds to several sites in the BES1 promoter 60
Figure 9. RAV1 binds to a specific site of the BZR1 promoter 62
Figure 10. The overexpression of RAV1 in bes1-D attenuated bes1-D phenotypes 66
Figure 11. The attenuation of bes1-D phenotypes by the overexpression of RAV1 was maintained continuously in the later stage of growth 68
Figure 12. Raw data of RNA-sequencing after normalization 74
Figure 13. Differentially regulated genes (DEGs) that were oppositely regulated by RAV1 and BES1 were identified by RNA-sequencing analysis 76
Figure 14. Quantitative RT-PCR analysis of target genes for RAV1 and BES1 in the RAV1-overexpressing transgenic plants 77
Figure 15. BAK1 interacts with PYR1 107
Figure 16. Subcellular localization of PYR1 and PYL4 109
Figure 17. BAK1 phosphorylates PYR1 on T137 and S142 sites 113
Figure 18. BAK1 specifically phosphorylates PYR1 116
Figure 19. Several conserved serine and threonine sites were considered as putative phosphorylation sites of PYR1 by BAK1 117
Figure 20. BAK1 interacts with and phosphorylates PYL4 119
Figure 21. PYR1DD and PYR1AA variants activate ABA-induced RD29B expression 124
Figure 22. BAK1-mediated phosphorylation of PYR1 increases leaf temperature 126
Figure 23. PYR1DD and PYR1AA variants increase ABA responses in plant 127
Figure 24. BAK1 phosphorylation of PYR1 or PYL4 increased ABA binding capacity 132
Figure 25. PYR1, PYR1DD, and PYR1AA were expressed in yeast 136
Figure 26. BAK1 phosphorylation of PYR1 resulted in reduced dimer formation of PYR1 137
Figure 27. BAK1 phosphorylation on PYR1 enhanced ABA-induced interactions between PYR1 and ABI1 142
Figure 28. Photographs of the second and third repeats of the results shown in Figure 1C (A), Figures 2E,G (B,C), and Figure 6B (D) 145
Figure 29. Updated model showing coordination of the initial stage of ABA signaling 154