Regulatory role of wheat E3 ligases in flowering time

다국어 초록 (Multilingual Abstract)

Hexaploid wheat (Triticum aestivum L.) developmental phase can be subdivided into three main phases, based on morphological changes of the SAM (Shoot Apical Meristem): vegetative phase (leaf initiation), pre-flowering reproductive phase (floret initiation and floral transition), and late pre-flowering reproductive phase (inflorescence growth). Floral transition, a phase change from the vegetative to the reproductive phase, is essential in plant organ development, and it mainly occurs when there is a change in the progress of the shoot in response to stimuli that promote reproductive development. In this regard, the reproductive phase initiates primordia development within the spikelet and determines spike fertility, which is a result of floret generation and degeneration. Flowering time determines reproductive success in plants and has a significant effect on yield in grain crops.
Ubiquitination acts as a post-translational modification, which mediates a variety of cellular function in eukaryotic cellular processes. In higher plants, ubiquitin-mediated protein regulation plays an important role in many aspects of plant developmental process, hormone, biotic and abiotic stress. In the ubiquitination pathway, E3 ligase regulates an essential role of the induced target substrate gene or protein level. E3 ligase, play a various role in the regulation of plant external and internal stimuli, including developmental growth. In this research, we aim to explore how RING and SCF type E3 ligases interact with and regulate substrate proteins involved in the flowering process.
In Chapter 1, floret primordia development rapidly reaches abortion stages after 50 d of vernalization treatment in the winter wheat varieties Keumgang and Yeongkwang. RNA-sequencing revealed a number of candidate genes that are differentially regulated during reproductive stages of primordia development, among which, we selected the RING-type E3 ligase TaBAH1 (TraesCS5B01G373000). TaBAH1 specifically induced from the double-ridge (WS2.5) to later stages (WS10) of floret primordium development and interacts with TaSAHH1. The overexpression of TaBAH1 in Arabidopsis showed early flowering phenotype under long-day condition. These results demonstrated that TaBAH1 positively regulates the flowering time via the regulation of the TaSAHH1 protein in wheat.
In Chapter 2, TaF-box3 (TraesCS6A01G407600) gene whose transcription level was significantly increased at the booting to anthesis (WS8 to 10) stages in wheat. The relative overexpression of TaF-box3 in Arabidopsis resulted in a markedly early flowering phenotype, which was associated with the repression of FLC levels and a reduction in H3K4me3 and H3K36me3 at the FLC locus. Furthermore, comparative proteomic 2DE analysis showed that the levels METS1 were significantly reduced in the TaF-box3 overexpression line. In this regard, TaF-box3 directly interacts with TaMETS1, thereby targeting it for ubiquitination and mediating the 26S proteasomal pathway. Our findings provide new insights into the mechanisms by which TaF-box3 is associated with histone modifications and flowering pathways.
In Chapter 3, TaFRFP (TraesCS1A02G143300) gene whose transcription level was highly increased at reproductive phase (WS2.5 to 9) in wheat. The relative overexpression of TaFRFP in Arabidopsis resulted in a significantly early flowering phenotype. TaFRFP overexpressing Arabidopsis plants also exhibit a hyposensitive phenotype with root growth and accumulate more SA contents. Furthermore, comparative proteomic 2DE analysis showed that the level of protein (FRL4A) were significantly reduced in TaFRFP overexpression plants under SA-treatment condition. Moreover, TaFRL4A is indispensable for FRL4A function in controlling flowering time and regulating FLC expression. Furthermore, we found that TaFRFP directly interacts with TaFRL (Arabidopsis FRL4A orthologues in wheat), thereby targeting it for ubiquitination and mediates 26S proteasomal pathway. Our findings provide new insights into the mechanisms by which TaFRFP is associated with the SA related flowering pathways.
In Chapter 4, TaFRFP2 (TraesCS2D01G516000) gene, whose transcription level was highly increased during the reproductive phase (WS2.5 to 9), was shown to induce a significantly early flowering phenotype when overexpressed in Arabidopsis. Moreover, TaFRFP2 is highly induced under salt stress and contribute to salt stress resistance in Arabidopsis. Comparative proteomic 2DE analysis revealed that the level of protein (LOX2) was significantly reduced in TaFRFP2 overexpression plants compared to WT. Furthermore, we found that TaFRFP2 directly interacts with TaLOX2 (the wheat orthologue of Arabidopsis LOX2), thereby targeting it for ubiquitination and mediating its degradation via 26S proteasomal pathway. Our findings provide new insights into the mechanisms by which TaFRFP2 is associated with flowering pathways.

목차 (Table of Contents)

• ABSTRACT i
• 국문 초록 viiv
• PREFACE vii
• ACKNOWLEDMENTS xviii
• TABLE OF CONTENTS ⅹ
• LIST OF TABLES ⅹxviii
• LIST OF FIGURES xix
• CHAPTER Ⅰ. Identification and analysis of a differentially expressed wheat RING-type E3 ligase in spike primordia development during post-vernalization xxi
• SYNOPSIS 2
• 1. INTRODUCTION 3
• 2. MATERIALS AND METHODS 7
• 2.1 Plant material and growth conditions 7
• 2.2 RNA-sequencing analysis 7
• 2.3 Gene cloning and vector construction 8
• 2.4 Gene expression studies 11
• 2.5 Subcellular localization 11
• 2.6 In vitro ubiquitination assay 12
• 2.7 Yeast two-hybrid assay 13
• 2.8 In vitro pull-down assay 14
• 2.9 In vitro cell-free degradation assay 14
• 2.10 Heterologous overexpression of TaBAH1 in Arabidopsis 14
• 3. RESULTS 16
• 3.1 Vernalization accelerates floral primordium development in wheat 16
• 3.2 Vernalization duration alters the time to heading in spring and winter
• wheat 16
• 3.3 Expression profiles of genes at different stages of floret development 20
• 3.4 TaBAH1 has E3 ligase activity 22
• 3.5 Wheat TaBAH1 interacts physically with TaSAHH1 24
• 3.6 TaBAH1 mediates TaSAHH1 polyubiquitination and proteasomal degradation 27
• 3.7 TaBAH1-overexpressing Arabidopsis represents a early-flowering phenotype 30
• 4. DISCUSSION 32
• 5. REFERENCES 38
• CHAPTER Ⅱ. The wheat TaF-box3, SCF ubiquitin ligase component, participates in the regulation of flowering time in transgenic Arabidopsis 43
• SYNOPSIS 44
• 1. INTRODUCTION 45
• 2. MATERIALS AND METHODS 48
• 2.1 Plant material and growth conditions 48
• 2.2 Gene expression 48
• 2.3 Subcellular localization 50
• 2.4 Gene sequence analysis 50
• 2.5 Yeast two-hybrid assay 50
• 2.6 Ubiquitination assay 50
• 2.7 Promoter Isolation and GUS expression 51
• 2.8 Plant transformation 51
• 2.9 Analysis of the leaf area and epidermal cell size 51
• 2.10 Histone modification analysis 52
• 2.11 2D PAGE and MALDI-TOF MS 52
• 2.12 Pull-down assay 53
• 2.13 Co-immunoprecipitation (Co-IP) assays 53
• 2.14 Protein degradation analysis 54
• 3. RESULTS 55
• 3.1 Expression analysis of TaF-box3 during floret development 55
• 3.2 SCFTaF-box3 possesses E3 ligase activity and Subcellular localization of
• TaF-box3 57
• 3.3 TaF-box3 interacts with TaSKPs 59
• 3.4 Expression Pattern of TaF-box3::GUS in Arabidopsis 59
• 3.5 Early-flowering of TaF-box3 overexpression in Arabidopsis 62
• 3.6 H3 methylation alterations in TaF-box3 62
• 3.7 Leaf cell growth of TaF-box3 66
• 3.8 2DE-PAGE and MALDI-TOF analysis in Transgenic Arabidopsis Plants 68
• 3.9 TaF-box3 directly interacts with TaMETS1 70
• 4. DISCUSSION 74
• 5. REFERENCES 78
• CHAPTER Ⅲ. The RING-type E3 ligase, TaFRFP, regulates flowering by controlling a salicylic acid-mediated floral promotion 87
• SYNOPSIS 85
• 1. INTRODUCTION 86
• 2. MATERIALS AND METHODS 89
• 2.1 Plant materials and experimental conditions 89
• 2.2 Gene expression 89
• 2.3 Subcellular localization 91
• 2.4 Gene sequence analysis 91
• 2.5 Yeast two-hybrid assay 91
• 2.6 Ubiquitination assay 91
• 2.7 Promoter Isolation and GUS expression 92
• 2.8 Plant transformation 92
• 2.9 ChIP assay 92
• 2.10 2D PAGE and MALDI-TOF MS 93
• 2.11 Co-immunoprecipitation (co-IP) assays 94
• 2.12 Protein degradation analysis 94
• 3. RESULTS 96
• 3.1 Expression analysis of TaFRFP in various tissues and during reproductive development stages 96
• 3.2 TaFRFP possesses E3 ligase activity and localized in nucleus 98
• 3.3 Expression Pattern of TaFRFPpro:GUS in Arabidopsis 100
• 3.4 Early-flowering of TaFRFP overexpression in Arabidopsis 102
• 3.5 TaFRFP involved in SA signaling 104
• 3.6 2DE-PAGE and MALDI-TOF analysis of TaFRFP transgenic in Arabidopsis 106
• 3.7 FRL4A represses flowering time in a TaFRFP-dependent manner 108
• 3.8 The enrichment of the active histone mark H3K4me3 is reduced at the FLC locus in the presence of TaFRFP 112
• 3.9 FRL4A negatively regulate SA signaling 112
• 3.10 TaFRFP directly interacts with TaFRL 116
• 3.11 TaFRFP mediates TaFRL through proteasomal degradation 118
• 4. DISCUSSION 120
• 5. REFERENCES 124
• CHAPTER Ⅳ. TaFRFP2: A wheat ring-type E3 ligase modulate flowering time and enhance salt stress tolerance in Arabidopsis 132
• SYNOPSIS 133
• 1. INTRODUCTION 134
• 2. MATERIALS AND METHODS 137
• 2.1 Plant materials and experimental conditions 137
• 2.2 Gene expression 137
• 2.3 Plant transformation 149
• 2.4 Phenotypic analysis of TaFRFP2 overexpression plant 149
• 2.5 Subcellular localization 140
• 2.6 Gene sequence analysis 140
• 2.7 Yeast two-hybrid assay 140
• 2.8 Ubiquitination assay 140
• 2.9 2D PAGE and MALDI-TOF MS 141
• 2.10 Protein degradation analysis 142
• 3. RESULTS 143
• 3.1 Expression analysis of TaFRFP2 in various tissues and reproductive developmental stages 143
• 3.2 GUS expression analysis in the TaFRFP2 transgenic Arabidopsis plant 143
• 3.3 Overexpression of TaFRFP2 induce early flowering in Arabidopsis 146
• 3.4 Overexpression of TaFRFP2 showed salinity tolerance in Arabidopsis 148
• 3.5 TaFRFP2 possesses E3 ligase activity and localized in nucleus 151
• 3.6 DE-PAGE and MALDI-TOF analysis of TaFRFP2 transgenic in Arabidopsis 153
• 3.7 TaFRFP2 directly interacts with TaLOX2 155
• 3.8 TaFRFP2 facilitates TaLOX2 degradation through proteasomal degradation 157
• 4. DISCUSSION 159
• 5. REFERENCES 165
• CHAPTER Ⅴ. CONCLUSION 170