Title Page
Abstract
Contents
Abbreviations 15
Chapter 1. C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 protein promotes flowering with TAF15b by repressing the floral repressor gene FLOWERING LOCUS C 18
Ⅰ. Introduction 19
Ⅱ. Materials and Methods 22
Plant materials 22
Plasmid construction and generation of transgenic lines 22
Growth conditions and flowering time measurement 23
RNA extraction and RT-qPCR analysis 23
Affinity purification followed by LC-MS/MS analysis 23
Y2H assay 24
Pull-down assay 25
Co-immunoprecipitation assay (CoIP) 25
Bimolecular fluorescence complementation analysis (BiFC) 25
ChIP assay 26
Immunoblotting 26
Library preparation and sequencing 27
Ⅲ. Results 31
CPL1 physically interacts with TAF15b 31
CPL1 promotes flowering 39
CPL1 reduces FLC expression 43
Effects of CPL1 on FLC chromatin 49
CPL1 reduces phosphorylated Pol II enrichment on FLC chromatin 53
TAF15b and CPL1 shares common target genes 56
Ⅳ. Discussion 61
Chapter 2. The two clock proteins CCA1 and LHY activate VIN3 transcription during vernalization through the vernalization-responsive cis-element 65
Ⅰ. Introduction 66
Ⅱ. Materials and Methods 71
Plant materials 71
Generation of transgenic lines 71
Growth conditions and treatments 72
GUS staining and MUG assay 73
RNA extraction and RT-qPCR analysis 73
Immunoblotting 73
EMSA 74
Y1H assay 75
Y2H assay 75
ChIP assay 76
Statistical analysis 76
Motif search 76
Ⅲ. Results 82
A proximal region in the VIN3 promoter is sufficient for the vernalization response 82
The promoter and first intron of VIN3 have distinct roles in VIN3 transcription 89
The G-box and EE in the VIN3 promoter are required for the vernalization response 94
CCA1 directly associates with VREVIN3 on the VIN3 promoter[이미지참조] 98
NTL8 does not associate with VREVIN3 or CCA1/LHY[이미지참조] 103
CCA1 and LHY rhythms are tuned to VIN3 expression pattern during vernalization 106
The effects of CCA1 and LHY on VIN3 induction are limited to the early stage of vernalization with near-freezing temperatures 115
CCA1 and LHY contribute to the vernalization-triggered VIN3 induction at mild cold temperatures 122
Ⅳ. Discussion 127
References 132
Abstract in Korean 149
Table 1. Primer sequences used in Chapter 1. 28
Table 2. Gene ontology enriched in common targets of TAF15b and CPL1. 59
Table 3. Primer sequences used in Chapter 2 78
Figure 1. Identification of CPL1 as a binding partner of TAF15b. 33
Figure 2. Yeast two-hybrid assay using TAF15b and CPL1. 34
Figure 3. Semi-in vivo pull-down assay between TAF15b and CPL1. 35
Figure 4. Co-immunoprecipitation (Co-IP) assay between TAF15b and CPL1 proteins expressed in Nicotiana benthamiana leaves. 36
Figure 5. Bimolecular fluorescence complementation (BiFC) analysis between TAF15b and CPL1. 37
Figure 6. Negative control of the BiFC between TAF15b and CPL1. 38
Figure 7. The flowering phenotype of cpl1 mutant. 40
Figure 8. Complementation analysis of CPL1. 41
Figure 9. Transcript levels of floral integrator genes in cpl1-7 mutants 42
Figure 10. Effect of cpl1 on the expression of floral repressor FLC. 44
Figure 11. Complementation analysis of CPL1. 45
Figure 12. Epistatic analysis between flc-3 and cpl1-7 46
Figure 13. Transcript levels of autonomous pathway genes in cpl1-7 mutants. 47
Figure 14. Relative transcript levels of FLC in CPL1 overexpressor. 48
Figure 15. CPL1 binds to FLC chromatin. 50
Figure 16. ChIP analysis showing the effect of cpl1-7 on TAF15b occupancy at the FLC promoter. 51
Figure 17. ChIP analysis of epigenetic markers at FLC chromatin. 52
Figure 18. Protein level of Pol II in cpl1-7, taf15b, and taf15b cpl1. 54
Figure 19. Increased phosphorylated-RNA polymerase II (Pol II) enrichment in cpl1. 55
Figure 20. RNA-seq and differentially expressed gene (DEG) analysis of cpl1-7 and taf15b-1. 57
Figure 21. Heat map representation of differentially expressed genes in cpl1-7 and taf15b-1, compared to Col-0. 58
Figure 22. The schematic working model how TAF15b-CPL1 complex promotes flowering by directly repressing FLC transcription. 64
Figure 23. Endogenous VIN3 expression and GUS activity of pVIN3_U_I:GUS. 84
Figure 24. Characterization of vernalization-responsive region in the VIN3 promoter. 86
Figure 25. Extent of conservation of the promoters of VIN3 orthologs in sister species of Arabidopsis thaliana. 87
Figure 26. Conservation of promoter sequences for VIN3 orthologs in Brassicaceae. 88
Figure 27. The 5'-UTR and the first intron of VIN3 has transcriptional enhancer activity independent of the vernalization response. 90
Figure 28. The first intron of VIN3 has transcriptional enhancer activity independent of the vernalization response. 92
Figure 29. Identification of vernalization-responsive cis-element in VIN3 on VIN3 promoter. 95
Figure 30. The flowering time of the FRI vin3-4 mutant transformed with the 3-kb pVIN3_U_I:VIN3 transgenes containing mutations in the G-box and/or EE motif. 97
Figure 31. The core circadian clock component CCA1 directly associates with the EE within VREVIN3 to regulate VIN3 expression.[이미지참조] 100
Figure 32. Chromatin immuno-precipitation (ChIP) experiments showing CCA1 occupancy across VIN3. 101
Figure 33. CCA1 protein directly binds to the VIN3 promoter. 102
Figure 34. NTL8 protein does not bind to VREVIN3.[이미지참조] 104
Figure 35. NTL8 protein does not interact with CCA1/LHY protein. 105
Figure 36. Diurnal rhythms of CCA1 and LHY are altered and CCA1 levels increase during vernalization. 108
Figure 37. Diurnal rhythms of CCA1 and LHY are rapidly altered after a few days of cold exposure. 109
Figure 38. Diurnal rhythms of CCA1 and LHY proteins, with or without 40 d of vernalization. 110
Figure 39. A biological replicate of daily rhythms of CCA1 and LHY proteins, with or without 40 days of vernalization. 111
Figure 40. Protein levels of CCA1 at ZT8 during vernalization. 112
Figure 41. A biological replicate of CCA1 protein levels at ZT8 during vernalization, as shown in Figure 40. 113
Figure 42. ChIP assay showing CCA1 enrichment at the VIN3 promoter region 114
Figure 43. Effects of CCA1 and/or LHY loss of function on the vernalization response upon 4℃ vernalization. 117
Figure 44. Effects of cca1 lhy on the VIN3 expression during the early phase of 4℃ vernalization. 118
Figure 45. Effects of CCA1 and/or LHY loss of function on the flowering time upon 4℃ vernalization. 119
Figure 46. VIN3 expression in CCA1- or LHY-overexpressing transgenic plants during 4℃ vernalization. 121
Figure 47. Effects of CCA1 and/or LHY loss of function on vernalization response upon 12℃ vernalization. 123
Figure 48. Effects of CCA1 and/or LHY loss of function on flowering time upon 12℃ vernalization. 124
Figure 49. A model for VIN3 regulation during vernalization. 126