E3 SUMO ligase AtSIZ1 regulates FLC-mediated flowering repression

다국어 초록 (Multilingual Abstract)

Flowering Locus C (FLC), a floral repressor, is antagonistic regulator for transition from vegetative to the reproductive phase. FLC represses expression of several genes involved in floral induction and its transcription is positively or negatively regulated by FRIGIDA or by vernalization, respectively. Although several factors affecting the transcription of FLC have been described, the post-translational regulation of FLC stability and function has not been clearly characterized. Here, I investigated the mechanisms regulating the activity and stability of the FLC protein.
Sumoylation, a post translational process of attaching small ubiquitin-related modifier (SUMO) to a lysine residue in proteins through an enzyme cascade catalyzed by E1, E2 and E3 enzymes, plays crucial role in protein stability and biological processes.
Bimolecular fluorescence complementation, co-immunoprecipitation and in vitro pull down analysis showed that FLC interacts with the E3 small ubiquitin-like modifier (SUMO) ligase AtSIZ1, suggesting that AtSIZ1 is an E3 SUMO ligase for FLC. In vitro sumoylation assays showed that FLC is modified by SUMO in presence of SUMO-activating enzyme E1 and conjugating enzyme E2 but its sumoylation is inhibited by AtSIZ1. In transgenic plants, inducible AtSIZ1 overexpression led to an increase in the concentration of FLC and delayed the post-translational decay of FLC, indicating that AtSIZ1 stabilizes FLC through direct binding. Also, the flowering time in mutant FLC (K154R, a mutation of sumoylation site)-overexpressing plants was comparable to that in wild type, whereas flowering was considerably delayed in FLC-overexpressing plants, supporting the notion that sumoylation is an important mechanism for FLC function. These results indicate that the sumoylation of FLC is critical for its role in the control flowering time and AtSIZ1 positively regulates FLC-mediated floral suppression.

목차 (Table of Contents)

• ABSTRACT Ⅰ
• CONTENTS Ⅲ
• LIST OF FIGURES Ⅵ
• LIST OF TABLES Ⅷ
• LIST OF ABBREVIATIONS Ⅸ
• LITERATURAL REVIEWS 1
• 1. SUMOylation 1
• 1.1. The SUMOylation pathway 1
• 1.2. Components of the SUMO pathway in Arabidopsis 3
• 2. Role of the SUMO in plants 6
• 2.1. Responses to abiotic stress 6
• 2.2. Responses to hormone signaling 9
• 2.3 Control of flowering 10
• 2.4. Defense reactions to pathogen attack 11
• 2.5. Metabolic regulation 12
• 3. Regulation of flowering time in Arabidopsis 14
• 3.1. Photoperiod pathway 14
• 3.2. Gibberellin pathway 15
• 3.3. Autonomous pathway 16
• 3.4. vernalization pathway 18
• INTRODUCTION 20
• MATERIALS AND METHODS 22
• 1. Plant materials and growth conditions 22
• 2. Construction of recombinant plasmids 22
• 3. Production of transgenic Arabidopsis plants 24
• 4. Purification of recombinant proteins 24
• 5. In vitro binding assay 25
• 6. In vivo Co-immunoprecipitation 27
• 7. Sumoylation assay 27
• 8. BiFC assay 28
• 9. Quantitative Real-Time RT-PCR analysis 29
• 10.β-estradiol and cycloheximide treatments 30
• 11. Cell-free degradation assay 30
• 12. Yeast two-hybrid assays 31
• 13. Investigation of flowering time 31
• RESULTS 35
• 1. AtSIZ1 physically interacts with FLC 35
• 2. FLC sumoylated without AtSIZ1 41
• 3. AtSIZ1 inhibits FLC sumoylation 44
• 4. Identification of sumoylation site on FLC 46
• 5. FLC stabilized by AtSIZ1 49
• 6. Modification by SUMO is necessary for flowering repression 57
• 7. Mutant FLC interacts with AtSIZ1 and FLC 62
• DISCUSSION 65
• REFERENCES 71
• ABSTRACT IN KOREAN 90