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Molecular genetic studies on the adaptive strategies of plants in changing environments : 환경 변화 속 식물의 적응 전략에 대한 분자 유전학적 연구
DC Field | Value | Language |
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dc.contributor.advisor | 박충모 | - |
dc.contributor.author | 박미정 | - |
dc.date.accessioned | 2017-07-14T05:55:25Z | - |
dc.date.available | 2017-07-14T05:55:25Z | - |
dc.date.issued | 2015-08 | - |
dc.identifier.other | 000000056977 | - |
dc.identifier.uri | https://hdl.handle.net/10371/125284 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 화학부 생화학전공, 2015. 8. 박충모. | - |
dc.description.abstract | Unlike animals, plants spend their entire lifetime in one position and are unable to escape from the unfavorable environmental conditions. As a consequence, plants have evolved diverse and effective strategies to monitor and adapt to various environmental conditions. For instance, it is well known that controlled cleavage of membrane-bound transcription factors ensures rapid transcriptional responses to abrupt environmental stresses in plants.
In this study, I investigated diverse molecular mechanisms allowing plants to cope with environmental changes. In Chapter 1, I examined the regulatory mechanism of the membrane-bound transcription factor (MTF). NTL6 is a plasma membrane-associated transcription factor and positively regulates drought resistance in Arabidopsis. I found that SnRK2.8 directly interacts with NTL6 in the cytoplasm. SnRK2.8 phosphorylates NTL6 primarily at Thr142 and SnRK2.8-mediated phosphorylation is required for the nuclear import of NTL6. Futhermore, the drought-resistant phenotype of 35S:NTL6 transgenic plants was compromised in 35S:NTL6 X snrk2.8-1 plants. These observations indicate that SnRK2.8-mediated protein phosphorylation, in addition to a proteolytic processing event, is required for NTL6 function in drought-stress signaling. In Chapter 2, I investigated the roles of BCD1 in iron homeostasis under osmotic stress. The BCD1 gene is regulated by the iron availability: induced by excessive iron, but repressed by iron deficiency. It is also induced under osmotic stress conditions such as high salinity and drought. Whereas the activation-tagged mutant bcd1-1D accumulated a lower amount of iron, the iron level was elevated in the knockout mutant bcd1-1. I also found that the BCD1 protein is localized to the Golgi complex. I propose that the BCD1 transporter plays a role in the iron homeostasis by reallocating excess iron released from the damaged cells exposed to osmotic stress. In the study of iron in plants, the conventional histochemical staining methods, such as Perls staining are still widely used. I also adopted Perls staining to determine the localization of iron in Arabidopsis in Chapter 2. However, it suffers from relatively poor resolution and detection limit. To improve the detection of iron in plants, in Chapter 3, I described a nobel method for high-sensitivity fluorescence imaging of iron, which demonstrates the amount and distribution of iron in plant tissues more precisely than conventional methods. Changes in day-length accompanied by seasonal changes are one of the major environmental factors that affect flowering time. In Arabidopsis, the diurnal control of CONSTANS (CO) accumulation by the circadian clock and light signals is critical for day-length measurement and therefore, for the photoperiodic flowering. While diverse molecular mechanisms are known to regulate the diurnal CO dynamics, it has never been explored whether and how CO itself contributes to this process. In Chapter 4, I demonstrated that CO undergoes alternative splicing, producing two protein isoforms, the full-size COa that is equivalent to the canonical CO transcription factor and the C-terminally truncated COb. Notably, I found that COb, which is resistant to the E3 enzymes, facilitates COa degradation by modulating the accessibility of COa to E3 ubiquitin ligases, providing a self-regulatory role of CO in its own diurnal dynamics. | - |
dc.description.tableofcontents | CONTENTS
ABSTRACT..................................................................................i CONTENTS................................................................................iii LIST OF FIGURES...................................................................ix LIST OF TABLES..................................................................xiii ABBREVIATIONS...................................................................xiv CHAPTER 1. Controlled nuclear import of NTL6 transcription factor reveals a cytoplasmic role of SnRK2.8 in drought stress response ABSTRACT..................................................................................2 INTRODUCTION........................................................................3 MATERIALS AND METHODS Plant materials and growth conditions....................................6 Drought-stress treatment........................................................6 Transcript level analysis........................................................6 Subcellular localization assays...............................................7 Preparation of recombinant proteins.......................................8 In vitro pull-down assays.....................................................8 Bimolecular fluorescence complementation (BiFC) assays........9 Transcriptional activation activity assays.................................9 In vivo phosphorylation assays............................................10 In vitro phosphorylation assays............................................10 Two-dimensional gel electrophoresis (2-DE) analysis.............10 RESULTS NTL6 promotes dehydration resistance.................................14 SnRK2.8 phosphorylates NTL6............................................19 Substitution of Thr142 reduces NTL6 phosphorylation............26 Thr142 phosphorylation is important for nuclear import..........32 SnRK2.8 phosphorylation of NTL6 contributes to drought resistance...........................................................................43 DISCUSSION.............................................................................47 CHAPTER 2. A Golgi-localized MATE transporter mediates iron homeostasis under osmotic stress in Arabidopsis ABSTRACT................................................................................53 INTRODUCTION......................................................................54 MATERIALS AND METHODS Plant materials and growth conditions..................................58 Treatments with growth hormones and abiotic stresses..........58 Analysis of transcript levels................................................59 Escherichia coli complementation assays..............................60 Histological assays..............................................................60 Measurements of chlorophyll content...................................60 Iron treatments...................................................................60 Perls iron staining..............................................................62 Measurement of iron content...............................................62 Subcellular localization assays.............................................63 RESULTS bcd1-1D mutant exhibits stunted growth and leaf chlorosis...65 BCD1 is a member of the MATE family............................69 BCD1 is induced by dark and abiotic stress........................74 BCD1 function is related to chlorosis..................................74 Expression of the BCD1 gene is regulated by iron availability .........................................................................................77 Iron content is lower in the bcd1-1D mutant.......................84 BCD1 protein is localized to the Golgi complex..................90 DISCUSSION Iron homeostasis and abiotic stress......................................95 Is BCD1 an iron transporter?..............................................97 CHAPTER 3. High-sensitivity fluorescence imaging of iron in plant tissues ABSTRACT..............................................................................101 INTRODUCTION....................................................................102 MATERIALS AND METHODS Synthesis of 7-(4-methylpiperazin-1-yl)-4-nitrobenzo-2-oxa-1,3- diazole (MPNBD).............................................................104 Metal ion sensing by MPNBD..........................................104 Plant materials and growth conditions................................105 Staining of iron in Arabidopsis plants................................105 Fluorescence microscopy...................................................106 RESULTS.................................................................................107 DISCUSSION...........................................................................130 CHAPTER 4. Self-directed control of the diurnal CONSTANS dynamics in Arabidopsis photoperiodic flowering ABSTRACT..............................................................................132 INTRODUCTION....................................................................133 MATERIALS AND METHODS Plant materials and growth conditions................................136 Gene expression analysis...................................................136 Absolute quantification of gene transcripts..........................137 Flowering time measurement..............................................138 Transcriptional activation activity assay...............................138 Yeast two-hybrid assay......................................................138 Yeast three-hybrid assay....................................................139 In vivo ubiquitination assay...............................................139 In vitro pull-down assay...................................................140 Bimolecular fluorescence complementation (BiFC) assay.....141 Protein stability assay.......................................................141 RESULTS CO alternative splicing and interactions between two CO isoforms...........................................................................144 COb-mediated attenuation of COa function in flowering induction...........................................................................151 Inhibition of COa DNA binding by COb..........................154 Differential protein stabilities of CO isoforms....................157 Facilitation of COa degradation by COb...........................160 Effects of COb on the interactions between COa and E3 ligases .......................................................................................162 DISCUSSION Regulation of the diurnal COa accumulation by COb........165 Active role of substrate in enzyme reactions.......................166 REFERENCES.........................................................................168 PUBLICATION LIST.............................................................197 ABSTRACT IN KOREAN.....................................................199 | - |
dc.format | application/pdf | - |
dc.format.extent | 14015284 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | membrane-bound transcription factor | - |
dc.subject | iron homeostasis | - |
dc.subject | fluorescent probe for iron detection | - |
dc.subject | photoperiodic flowering | - |
dc.subject | alternative splicing | - |
dc.subject.ddc | 540 | - |
dc.title | Molecular genetic studies on the adaptive strategies of plants in changing environments | - |
dc.title.alternative | 환경 변화 속 식물의 적응 전략에 대한 분자 유전학적 연구 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Mi-Jeong Park | - |
dc.description.degree | Doctor | - |
dc.citation.pages | xv, 200 | - |
dc.contributor.affiliation | 자연과학대학 화학부 | - |
dc.date.awarded | 2015-08 | - |
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