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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | Florence Vignols(Florence Vignols),靳宗洛(Tsung-Luo Jinn) | |
dc.contributor.author | Hui-Chen Wu | en |
dc.contributor.author | 吳慧珍 | zh_TW |
dc.date.accessioned | 2021-06-15T07:04:19Z | - |
dc.date.available | 2013-02-09 | |
dc.date.copyright | 2011-02-09 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-12-27 | |
dc.identifier.citation | Ahn SG and Thiele DJ (2003) Redox regulation of mammalian heat shock factor 1 is essential for Hsp gene activation and protection from stress. Genes and Development 17: 516–528.
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Wu HC, Hsu SF, Luo DL, Chen SJ, Huang WD, Lur HS and Jinn TL (2010) Recovery of heat shock-triggered released apoplastic Ca2+ accompanied by pectin methylesterase activity is required for thermotolerance in soybean seedlings. J Exp Bot 61: 2843– 2852. Yang T and Poovaiah BW (2002) Hydrogen peroxide homeostasis: activation of plant catalase by calcium/ calmodulin. Proc Natl Acad Sci USA 99: 4097–4102. Zhong M, Orosz A and Wu C (1998) Direct sensing of heat and oxidation by Drosophila heat shock transcription factor. Molecular Cell 2: 101–108. Zou J, Guo Y, Guettouche T, Smith DF and Voellmy R (1998) Repression of heat shock transcription factor HSF1 activation by Hsp90 (Hsp90 complex) that forms a stress-sensitive complex with HSF1. Cell 94: 471–480. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48606 | - |
dc.description.abstract | 植物生活史中常遭遇環境之脅迫,如熱、乾旱及鹽害等逆境,當其改變幅度超過適合植物正常生命活動範圍時,對其生命活動則造成不利之影響。尤其以溫度增加所引起之熱逆境,為普遍存在的農業問題,導致作物產量大幅的削減。另外,熱逆境會誘導活性氧大量產生,造成植物體內氧化逆境之形成;因此,活性氧之清除也是避免細胞在熱逆境下受到傷害之重要機制。探討逆境信號在植物體內傳導途徑及基因表達調控等分子層次上之調節,可提供具潛力發展之基因工程策略,以改進植物對逆境之耐性,在理論及生產上提供重要之研究基礎與實踐意義。
博士論文第一部分,主要以水稻及大豆為模式植物探討「熱逆境誘導外源鈣離子與細胞壁果膠甲基酯酶,參與植物細胞壁重建及訊息傳導之調控」。植物能大量累積低分子量熱休克蛋白質(small HSP, sHSP),扮演著抗熱逆境之重要角色;然本研究發現植物在熱休克 (heat shock, HS) 誘導下,藉由細胞壁果膠甲基酯酶 (pectin methylesterase, PME) 活性之調節,及激發細胞壁結構性鈣之移動,一面參與細胞壁之重建,增強細胞壁結構及細胞間之黏結作用;另一面,誘導外源鈣離子進入細胞質中,提高鈣訊號之震盪幅度及頻率,由鈣調蛋白 (calmodium, CaM) 接收並將信息傳達至下游,誘導低分子量熱休克蛋白質之表現,而提升植物抗熱逆境之能力。 論文第二部分以阿拉伯芥為模式植物,探討「硫氧還蛋白與分子伴護蛋白系統之功能性基因體與蛋白質體之交互作用」。植物中特有的類硫氧還蛋白(thioredoxin-like)稱Tetratricoredoxin (TDX),兼具氧化還原活性中心及分子伴護之特性。利用酵母菌雙雜交系統 (Y2H) 及雙分子螢光互補技術 (BiFC),在活细胞內證實TDX與阿拉伯芥高分子量熱休克蛋白質70 (HSP70) 發生交互作用,推測TDX能夠穩定HSP70與基質之結合,間接參與變性蛋白構型及活性之恢復。並且TDX受氧化逆境之誘導,轉移並累積至細胞核中,推測其功能與氧化逆境訊息之傳遞相關;藉由TDX基因缺失突變株,對氧化及熱逆境敏感性下降之外表型,並提高逆境相關基因之表現,推測TDX位於逆境訊號路徑之上游,扮演著訊號接受的角色,此過程可能與HSP70間之交互作用有關。此研究首次發現植物硫氧還蛋白在氧化與熱逆境中,同時參與訊息傳遞及伴護蛋白之功能,對於蛋白質在逆境間之交互作用有實質的貢獻。 | zh_TW |
dc.description.abstract | While being unable to escape their lands, plants are continuously submitted to the modifications of their environment, and need to adjust proper physiological processes in response to various stimuli. During this work, I devoted my studies on two major stresses affecting plant development, heat shock (HS) and oxidative stresses (OS), focusing on key elements in these pathways (HS chaperons and HS-related thioredoxins) in order to bring news elements of knowledge and interconnexion of these pathways.
Using rice and soybean as mono- and dicotyledonous plant systems, I show how HS leads to calcium release from plant cell apoplast to the cytosol in a typical “calcium signature”, conferring cell wall rigidity and enhancing HS signaling pathway. I also identify pectin methylesterase (PME) as required in this pathway for cell wall remodeling and plasma membrane integrity. I further investigate how plant sense temperature increases and how they transmit the HS signal to downstream elements. Using systematic analyses of calmodulin (CaM) and small heat shock protein (sHSP) gene expression, I identify one CaM as a coordinator of HS response, which I characterize as involving specific cytosolic/nuclear isoforms of the sHSP family. I latter perform the molecular analysis of TDX, a thioredoxin suspected to be involved in heat shock response. I show that TDX interacts with nucleo-cytoplasmic members of the HSP70 family in a redox dependent manner, both HS and OS inducing its nuclear relocation, and that TDX is required for both acquired thermotolerance and OS signaling. I finally discuss the data brought by this work and propose models with cross-talks between HS and OS signaling. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T07:04:19Z (GMT). No. of bitstreams: 1 ntu-99-D93b42002-1.pdf: 28258892 bytes, checksum: f4bcff3fb5596157b987b9d2ec502c91 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | ABSTRACT 1
摘要 1 Summary 2 Resume 3 I- Chapter 1: Literature Review 4 I.1- Introduction 4 I.2 Components of Induced-Stress Tolerance 5 I.2.1-The Versatile Role of Calcium Ions (Ca2+) 6 I.2.1.1-Ca2+ Signature 6 I.2.1.2-Ca2+, CaM, and Heat Stress 7 I.2.2-The Role of Ca2+/Pectate Interaction for Cell Wall Remodeling to Confer Thermotolerance in Plants 8 I.2.2.1-Elements of Plant Cell Wall 8 I.2.2.2-Ca2+/Pectate Network 9 I.2.2.3-Apoplastic Calcium 10 I.2.2.4-Pectin Methylesterases 10 I.2.3-Overview of Heat-Shock Response and Stress Proteins 15 I.2.3.1-Chaperone 15 I.2.3.2-Heat Shock Protein 70 16 I.2.3.3-Small Heat Shock Proteins 17 I.2.4- Reactive Oxygen Species and Oxidative Stress 25 I.2.4.1-Production of ROS 25 I.2.4.2-Hydrogen Peroxide (H2O2) 26 I.2.4.5-ROS Scavenging Mechanisms in Cells 27 I.3-Simultaneous Activation of Heat Shock Response and Oxidative Stress 30 I.4-Redox Regulation-Reduction/Oxidation (Redox) Status Regulates in Various Aspects of Cellular Function 32 I.4.1-Thioredoxins 33 I.4.2- Features of Plant Thioredoxin 33 I.5-New Perspectives on Heat Shock Response in Plant 38 I.5.1-Thioredoxin and Chaperone Regulation 38 I.5.2-Tetratricoredoxin (TDX) 39 I.6- Concluding Remarks 44 I.7-The Aim of Project 44 I.8-REFERENCES 48 II-Chapter 2: The Crosstalk between Extracellular and Intracellular Calcium Mobilization in Cell Wall Remodeling and Heat Shock Signaling 58 II-Chapter 2-Part 1: Heat Shock-Triggered Released Apoplastic Ca2+ Accompanied by Pectin Methylesterase Activity and Cytosolic Ca2+Oscillation Are Crucial for Plant Thermotolerance 58 II.1.1 ABSTRACT 58 II.1.2-RESULTS 59 II.1.2.1-Conditions for Lethal Treatment and Thermotolerance Establishment in Rice 59 II.1.2.2-Effect of EGTA Treatment on Plant Growth and Cellular Leakage 60 II.1.2.3-Effect of HS-Released Ca2+ Concentration and Its Recovery on the Development of Thermotolerance 61 II.1.2.4-Effect of EGTA on sHSP Accumulation and Organelle Localization in Vivo and Thermostabilization of Soluble Proteins in Vitro 70 II.1.2.5-Time-Course Study of the [Ca 2+]cyt Oscillation during Heat Shock Response in Rice Root 73 II.1.2.6-Effect of HS and EGTA Treatment on Pectin Methylesterase (PME) and Polygalacturonase (PG) Activity 73 II.1.2.7-Status of Demethylesterified Pectin in Response to HS and EGTA Treatment 74 II.1.2.8-The “egg box” Model Structure, Ca2+-Demethylated Homogalacturonan, in Response to HS and EGTA treatment 75 II.1.3-DISCUSSION 84 II.1.3.1-Effect of EGTA on Thermotolerance 84 II.1.3.2-Cytosolic Ca2+ Oscillation during HS and EGTA Treatment 84 II.1.3.3-Ca2+-Pectate Enriched the Structure and PME Physiological Functions during HS and EGTA Treatment 85 II.1.4- MATERIALS AND METHODS 90 II.1.4.1-Plant Growth and Cellular Leakage Analysis 90 II.1.4.2-Post-Ribosomal Supernatant (PRS) Preparation and Fractionation 90 II.1.4.3-Assay for Thermal Denaturation of Soluble Proteins 90 II.1.4.4-Quantitation of Class-I Small Heat Shock Protein (sHSP) Levels 90 II.1.4.5-Ion Analysis 91 II.1.4.6-Pectin Methylesterase (PME) Activity Analysed by Acidic Continuous Native-PAGE 91 II.1.4.7-Polygalacturonase (PG) Activity Assay 91 II.1.4.8-Histochemical Analysis of Pectin by Ruthenium Red (RR) Staining 92 II.1.4.9-Immunolocalization of Ca2+-Demethylated Homogalacturonan 92 II.1.4.10-Statistical Analysis 93 II.1.5- REFERANCES 94 II-Chapter 2-Part 2: Calcium/Calmodium Is Critical for Heat Shock Signal Transduction in Rice 97 II.2.1-ABSTRACT 97 II.2.2-RESULTS 97 II.2.2.1-Changes of Cytosolic Ca2+ Concentration Occur in Rice during Heat Shock 97 II.2.2.2-Apoplast Mediates Heat Shock-Induced Calcium Entry in Rice Cytosol .. 98 II.2.2.3-Rice Calmodulins Are Involved in Heat Shock-Induced Calcium Entry in the Cytosol 100 II.2.2.4-Rice Calmodulin Genes Differentially Respond to Heat Shock upon Time 107 II.2.2.5-Nucleo-Cytoplasmic Small HSP Gene Expression Differentially Accompanies Calmodulin Gene Induction during Early Heat Shock Response 108 II.2.2.6-Apoplastic Ca2+ as a Source for Rice Calmodulin and Nucleo-Cytoplasmic Small HSP Gene Induction under Early Heat Shock 113 II.2.2.7-Subcellular Redistribution of OsCaM1-1 in Response to Heat Shock 122 II.2.2.8-Overexpression of OsCaM1-1 Induces Heat Shock-Related Gene Expression and Enhances Thermotolerance in Arabidopsis 126 II.2.3-DISCUSSION 131 II.2.3.1-In Rice, Apoplast Mediates Heat Shock-Induced Calcium Entry into Cytosol Following Typical Ca2+ Signature Dynamics 131 II.2.3.2-The Spatio-Temporal Effects of [Ca 2+]cyt Oscillation on Calmodulin and Small HSP Genes Expression during HS 132 II.2.3.3-Subcellular Redistribution of OsCaM1-1 in Response to Heat Shock 136 II.2.3.4-Constitutive OsCaM1-1 Expression Enhances the Thermotolerance in Arabidopsis 137 II.2.4-MATERIALS AND METHODS 140 II.2.4.1-Plant Materials 140 II.2.4.2-Study of the Kinetic Changes in Ca2+ Oscillation 140 II.2.4.3-RNA Extraction and RT-PCR Analyses 140 II.2.4.4-Subcellular Localization of OsCaM1-1-GFP Fusion Protein 141 II.2.4.5-Production of OsCaM1-1 Overexpressing Line in Arabidopsis 141 II.2.4.6-Thermotolerance Tests 142 II.2.4.7-Cellular Ion Leakage Analysis 142 II.2.4.8-SDS-PAGE and Western Blot Analysis 142 II.2.4.9-Statistical Analysis 143 II.2.5-REFERENCES 144 III-Chapter 3: Redox and Chaperone Net Work in Arabidopsis-Search for the Function of Tetratricoredoxin (TDX) during Oxidative and Heat Stresses 150 III-SUMMARY 150 III-Chapter 3-Part 1: Tetratricoredoxin (TDX) and the Closely Related HSP70-Interacting Protein (HIP) Differentially Sense Oxidative Stress in Arabidopsis 151 III.1.1-ABSTRACT 151 III.1.2-RESULTS 151 Section I: Regulation of TDX Nuclear Relocalization in Response to Oxidative Sress 151 SI.1-TDX and HIP Preferentially Accumulate in Developing Arabidopsis Tissues 151 SI.2-TDX and HIP Genes Are Differently Expressed under Oxidative and Temperature stresses 152 SI.3-TDX and Hip Reside in the Cytosol and in the Nucleus in Vivo 152 SI.4-Oxidative Stress Induces Stable Relocation of TDX but Not HIP into the Nucleus. 153 SI.5-Validation of TDX Nuclear Relocation under Oxidative Stress by Immuno Blotting Sub-Cellular Fractions 153 SI.6-The Truncation of the Thioredoxin Active Site Specifically Attenuates TDX Nuclear Relocation under Oxidative Stress 154 Section II: TDX Plays a Specific Role in Oxidative Stress Signal Transduction 164 SII.1-The tdx Mutants Show Lower Sensitivity towards Oxidative Stress 164 SII.2-tdx-Deficient Plants Accumulate Less H2O2 under Oxidative Stress 165 SII.3-tdx-Deficient Plants Exhibit Alteration of H2O2 Detoxification Systems 165 SII.4-tdx-Deficient Plants are not Altered in Superoxide Production 166 SII.5-Search for Modification of Oxidative Stress-Related Gene expression in tdx Mutant 166 SII.6-A hip Mutant also Shows Lower Sensitivity towards H2O2 but Does Not Show Defect in H2O2 and O2•– Accumulations 168 Section III: Search for TDX and HIP Functions in Additional Pathways 178 SIII.1-The tdx mutant but Not hip Is Insensitive to ABA 178 SIII.2-The hip.tdx Double Mutant Exhibit Enhanced Tolerance to Abiotic Stresses 178 SIII.3-A hip.tdx Double Mutant Is Sensitive to a Protein Synthesis Inhibitor 182 III.1.3-CONCLUSION AND PERSPECTIVES 184 III.1.4-MATERIAL AND METHODS 187 III.1.4.1-Plant Material and Growth Conditions 187 III.1.4.2-Identification of TDX and AtHIP Insertion Mutant Lines 187 III.1.4.3-RNA Extraction, RT-PCR and qPCR Analysis 188 III.1.4.4-Construction of Promoter TDX::GUS and HIP::GUS Fusion Genes 189 III.1.4.5-Fusion Constructs for Stable Expression in Arabidopsis 189 III.1.4.6-Histochemical GUS Staining 189 III.1.4.7-Gene Cloning and Subcellular Localization of GFP-Fusion Proteins 190 III.1.4.8-Preparation of the Cytosolic and Nuclear Proteins and Immunoblotting 191 III.1.4.9-Complementation Test 192 III.1.4.10-Stress Response Assays 193 III.1.4.11-Histochemical Detection of H2O2 and Superoxide Anions in Arabidopsis Leaves 193 III.1.4-REFERENCES 195 III.2-Chapter 3-Part 2: Tetratricoredoxin Is a HSP70-Interacting Protein Involved in Acquired Thermotolerance in Arabidopsis 199 III.2.1-ABSTRACT 200 III.2.2-RESULTS 200 Section I: The Partnership of TDX and HSP70s Family in Arabidopsis 200 SI.1-TDX Interacts Specifically with Arabidopsis HSP70sC/N Isoforms in Yeast Cells .200 SI.2-TDX-HSP70sC/N Interaction Efficiently Reports in Y2H Assay Involving Stress Responses 201 SI.3-TDX Interacts with HSP70sC/N in Planta ..202 SI.4-Search for Determinants in TDX Required for Interaction with HSP70C/N 213 SI.4.1- Both the TPR Motif and the Thioredoxin Domain of TDX Are Essential for Interacting with all HSP70sC/N 213 SI.4.2-The First Cys-304 of TDX Redox Center Is Involved in the Interaction with HSP70sC/N 213 SI.5-Both ATPase and Peptide Binding Domain of HSP70-1 Are Required for TDX Interaction 213 SI.6-Mutational Analysis of HSP70-1 214 Section II: HIP: another putative HSP70-interacting protein linked to TDX/HSP70s partnership 220 SII.1-HIP Interacts with HSP70sC/N in Yeast and Plant in Vivo Reporter Systems 220 SII.2-HIP Binds HSP70sC/N through Its N-terminal Domain, Displaying the Similar Pattern to TDX Binding 224 SII.3-Other Heat Shock Proteins Interact with TDX and HIP 227 SII.4-Dimerization Properties of TDX and HIP 229 Section III: TDX and HIP Are Components of Signaling Pathways with Similar Behaviors towards Heat Stress 231 SIII.1-Comparisons of HIP and TDX Gene Expression Patterns under Heat Stresses 231 SIII.2-Heat Shock Induces Transient Relocation of Both TDX and HIP into the Nucleus 232 SIII.3-The hip.tdx Double Mutant Exhibit Enhanced Tolerance to Heat Stress 232 SIII.4-The Transcript Level of Heat Related Gene Expression in TDX and HIP Mutant Background 233 III.2.3-DISCUSSION 238 III.2.3.1-TDX: An Atypical Thioredoxin Unifying Molecular Stress-Linked Chaperones Interactomes 238 III.2.3.2-TDX Interacts with Arabidopsis HSP70sC/N in Complex Chaperone Networks Probably Involving Several Classes of Heat Shock Proteins 239 III.2.3.3-Domain Mapping Highlights Specific Chaperone- and Redox-Linked Functions Required in TDX/HIP/HSP Networks 240 III.2.3.4-TDX and HIP Have Associated Function to Acquired Thermotolerance Process 241 III.2.4-MATERIALS AND METHODS 242 III.2.4-REFERENCES 243 III.3-Chapter 3-Part 3: Searching for TDX-Interacting Proteins: Characterization of a TDX-NAD Kinase Interaction 247 III.3.1-ABSTRACT 247 III.3.2-RESULTS 248 III.3.2.1-Identification of TDX Putative Targets by the Y2H Method 248 III.3.2.2-TDX Putative Target Proteins Are Involved in Distinct Pathways 248 III.3.2.3-TDX/NAD Kinase Interaction: a New Role of TDX in Regulating NADPH Pathway 253 III.3.2.3a-The NAD Kinase Family in Arabidopsis 253 III.3.2.3b-NADK1 and NADK2, but Not NADK3 Interact with TDX in a Y2H System.. 254 III.3.2.3c-TDX Interacts with NADK1 and NADK2 in Planta 254 III.3.2.3d-Are NADK Proteins Targeted by Other Redoxins or by HIP? 256 III.3.2.4-TDX Also Interacts with Distinct Proteins Involved in Different Pathways 265 III.3.3-CONCLUSIONS AND FUTURE PROSPECTS 270 III.3.4-MATERIALS AND METHODS (common to Chapter 3-Part 2+3) 274 III.3.4.1-Yeast Strains and Media 274 III.3.4.2-Two-Hybrid Experiments 274 III.3.4.3-DNA Cloning for Y2H Assay 275 III.3.4.4-Bimolecular Fluorescence Complementation (BiFC) Assay 275 III.3.4.5-RNA Extraction, RT-PCR and qPCR Analysis 276 III.3.4.6-Plant Material, Growth Conditions, and Mutants Identification 276 III.3.4.7-Thermotolerance Test 277 III.3.5-REFERENCES 278 IV-Chapter 4-Conclusions and Prospects 282 IV.1-Recovery of Heat Shock-Triggered Released Apoplastic Ca2+ Accompanied by Pectin Methylesterase Activity Is Required for Thermotolerance 282 IV.2-Early Heat Shock Signal Transduction Mechanisms: Newly Discovered Components Linked to Plant Thermotolerance 285 IV.3.3-The Hypothesis Model of TDX and HIP Function in Acquired Thermotolerance…. 290 IV.3.1-TDX May Serve as a Sensor/Transducer of Oxidative Stress Signals 290 IV.3.2-TDX/HSP70/HIP Interactome in Arabidopsis 292 IV.3.3-The Hypothesis Model of TDX and HIP Function in Acquired Thermotolerance 292 IV.4-Overall Comments 300 | |
dc.language.iso | en | |
dc.title | 植物熱休克反應之分子基礎研究-熱休克訊息傳遞相關分子之鑑定與氧化逆境交叉路徑之探討 | zh_TW |
dc.title | Molecular Bases of the Heat Shock Response in Plants- Identification of Elements Involved in Heat Shock Transduction Pathway and in the Cross Talk between Heat Shock and Oxidative Stress | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-1 | |
dc.description.degree | 博士 | |
dc.contributor.advisor-orcid | ,靳宗洛(jinnt@ntu.edu.tw) | |
dc.contributor.oralexamcommittee | Christophe Brugidou(Christophe Brugidou),林彩雲(Tsai-Yun Lin),林秋榮(Chu-Yung Lin),常怡雍(Yee-Yung Charng),葉開溫(Kai-Wun Yeh),林讚標(Tsan-Piao Lin) | |
dc.subject.keyword | 二價鈣離子,分子伴護蛋白,細胞壁果膠甲基酯酶,氧化逆境,硫氧還蛋白,鈣調蛋白,熱休克蛋白質,熱逆境, | zh_TW |
dc.subject.keyword | Calcium,Chaperone,Calmodium,Heat shock proteins,Oxidative stress,Pectin methylesterase,Tetratricoredoxin, | en |
dc.relation.page | 332 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-12-28 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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