臺灣博碩士論文加值系統

熱休克反應，為生物體內普遍存在的機制；藉由誘導熱休克蛋白質的生成，進行細胞保護，而熱休克蛋白質的生成是經由熱休克轉錄因子所調控。人類熱休克轉錄因子結合蛋白(HsHSBP1)可以和熱休克轉錄因子進行交互作用，終止熱休克反應，扮演負向調控者的角色。關於植物HSBP參與熱休克反應的機制目前並不清楚。藉由序列分析，我們在阿拉伯芥中篩選到一個HSBP基因(AtHSBP)，並分離出兩個基因缺陷突變株。經由測試其熱耐受性，顯示突變株具有較高的「誘導耐熱性」，但是對於「先天耐熱性」並沒有顯著的影響。在阿拉伯芥原生質體中表現GFP-AtHSBP，得知在正常生長情況以及熱處理的初期，AtHSBP表現於細胞質中；但長時間的熱處理及當其回復到正常生長情況時，AtHSBP會自細胞質轉移至細胞核。雙分子螢光互補(Bimolecular fluorescence complementation)分析的結果顯示，AtHSBP會和AtHSFA1a, AtHSFA1b以及AtHSFA2進行交互作用。利用即時定量聚合酶鍊鎖反應(quantitative real-time PCR)分析，得知AtHSBP缺陷突變株在遭受熱逆境時，熱休克蛋白基因(HSP101, HSP70, HSP17.4, HSP18.2, and HSP25.3)的表現量及累積量比野生型多；而AtHSBP的過度表現株，其熱休克蛋白基因的表現量及累積量下降，並導致其「誘導耐熱性」能力降低。經由以上結果，我們推測AtHSBP在熱休克反應對於熱休克蛋白的表現，扮演負向調控者的角色。

Abstract in Chinese---------------------------------------------------------------------------- 1
Abstract in English---------------------------------------------------------------------------- 2
Abbreviations----------------------------------------------------------------------------------- 3

Introduction------------------------------------------------------------------------------------- 4
Heat shock response and heat shock proteins----------------------------------------------- 4
Regulation of heat shock response ----------------------------------------------------------- 5
Heat shock factors in Arabidopsis------------------------------------------------------------ 6
Previous study of heat shock factor binding proteins-------------------------------------- 8
Goals and strategies of the dissertation------------------------------------------------------ 9

Materials and Methods---------------------------------------------------------------------- 11
Plant material, transformation and growth condition ------------------------------------- 11
RNA extraction and quantitative real-time PCR ------------------------------------------ 11
Thermotolerance phenotypes assay --------------------------------------------------------- 12
Transient expression in Arabidopsis protoplast-------------------------------------------- 12
Subcellular localization and bimolecular fluorescence complementation assay ------13

Results------------------------------------------------------------------------------------------ 14
Sequence alignment and phylogenetic analysis of the HSBP homologs across plants and animals------------------------------------------------------------------------------------- 14
Expression profiles of AtHSBP in various tissues and with HS treatment ------------ 15
Thermotolerance analysis in AtHSBP mutants-------------------------------------------- 15
AtHSBP is a cytosolic-localized protein capable of entering nucleus in response to thermal stress----------------------------------------------------------------------------------- 16
AtHSBP interacts with AtHSFA1a, AtHSFA1b and AtHSFA2 in vivo----------------- 17
The HSP expression patterns in AtHSBP mutants during HSR------------------------- 18

Discussion-------------------------------------------------------------------------------------- 19
AtHSBP is a conserved protein in HSBP family and may participate in embryogenesis and environmental stress---------------------------------------------------------------------- 19
AtHSBP acts as a negative regulator during HSR----------------------------------------- 20
AtHSBP participates in the temporal and spatial regulation of HSP genes expression under the influence of heat stress------------------------------------------------------------ 22

Figures and Table----------------------------------------------------------------------------- 23
References-------------------------------------------------------------------------------------- 35
Supplemental data---------------------------------------------------------------------------- 40

Abravaya K, Myers MP, Murphy SP, Morimoto RI (1992) The human heat-shock protein hsp70 interacts with hsf, the transcription factor that regulates heat-shock gene-expression. Genes & Development 6: 1153-1164
Alfonso M, Yruela I, Almarcegui S, Torrado E, Perez MA, Picorel R (2001) Unusual tolerance to high temperatures in a new herbicide-resistant D1 mutant from Glycine max (L.) Merr. cell cultures deficient in fatty acid desaturation. Planta 212: 573-582
Baler R, Welch WJ, Voellmy R (1992) Heat-shock gene-regulation by nascent polypeptides and denatured proteins - hsp70 as a potential autoregulatory factor. Journal of Cell Biology 117: 1151-1159
Balogi Z, Torok Z, Balogh G, Josvay K, Shigapova N, Vierling E, Vigh L, Horvath L (2005) "Heat shock lipid" in cyanobacteria during heat/light-acclimation. Archives of Biochemistry and Biophysics 436: 346-354
Baniwal SK, Chan KY, Scharf KD, Nover L (2007) Role of heat stress transcription factor HsfA5 as specific repressor of HsfA4. Journal of Biological Chemistry 282: 3605-3613
Boyer JS (1982) Plant productivity and environment. Science 218: 443-448
Busch W, Wunderlich M, Schoffl F (2005) Identification of novel heat shock factor-dependent genes and biochemical pathways in Arabidopsis thaliana. Plant Journal 41: 1-14
Charng YY, Liu HC, Liu NY, Chi WT, Wang CN, Chang SH, Wang TT (2007) A heat-inducible transcription factor, HsfA2, is required for extension of acquired thermotolerance in Arabidopsis. Plant Physiology 143: 251-262
Clarke SM, Mur LAJ, Wood JE, Scott IM (2004) Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana. Plant Journal 38: 432-447
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16: 735-743
Craig EA, Weissman JS, Horwich AL (1994) Heat-shock proteins and molecular chaperones - mediators of protein conformation and turnover in the cell. Cell 78: 365-372
Dat JF, Lopez-Delgado H, Foyer CH, Scott IM (1998) Parallel changes in H2O2 and catalase during thermotolerance induced by salicylic acid or heat acclimation in mustard seedlings. Plant Physiology 116: 1351-1357
Davletova S, Rizhsky L, Liang HJ, Zhong SQ, Oliver DJ, Coutu J, Shulaev V, Schlauch K, Mittler R (2005) Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 17: 268-281
Doering P, Treuter E, Kistner C, Lyck R, Chen A, Nover L (2000) The role of AHA motifs in the activator function of tomato heat stress transcription factors HsfA1 and HsfA2. Plant Cell 12: 265-278
Frydman J (2001) Folding of newly translated proteins in vivo: The role of molecular chaperones. Annual Review of Biochemistry 70: 603-647
Fu S, Rogowsky P, Nover L, Scanlon MJ (2006) The maize heat shock factor-binding protein paralogs EMP2 and HSBP2 interact non-redundantly with specific heat shock factors. Planta 224: 42-52
Fu SN, Meeley R, Scanlon MJ (2002) empty pericarp2 encodes a negative regulator of the heat shock response and is required for maize embryogenesis. Plant Cell 14: 3119-3132
Fu SN, Scanlon MJ (2004) Clonal mosaic analysis of EMPTY PERICARP2 reveals nonredundant functions of the duplicated HEAT SHOCK FACTOR BINDING PROTEINs during maize shoot development. Genetics 167: 1381-1394
Hancock KR, Phillips LD, White DWR, Ealing PM (1997) pPE1000: A versatile vector for the expression of epitope-tagged foreign proteins in transgenic plants. Biotechniques 22: 861-&
Hong SW, Vierling E (2000) Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress. Proceedings of the National Academy of Sciences of the United States of America 97: 4392-4397
Johnson JL, Craig EA (1997) Protein folding in vivo: Unraveling complex pathways. Cell 90: 201-204
Kim SY, Sharma S, Hoskins JR, Wickner S (2002) Interaction of the DnaK and DnaJ chaperone system with a native substrate, p1 RepA. Journal of Biological Chemistry 277: 44778-44783
Kotak S, Larkindale J, Lee U, von Koskull-Doring P, Vierling E, Scharf KD (2007) Complexity of the heat stress response in plants. Current Opinion in Plant Biology 10: 310-316
Kotak S, Port M, Ganguli A, Bicker F, von Koskull-Doring P (2004) Characterization of C-terminal domains of Arabidopsis heat stress transcription factors (Hsfs) and identification of a new signature combination of plant class A Hsfs with AHA and NES motifs essential for activator function and intracellular localization. Plant Journal 39: 98-112
Krishna P (2003) Plant responses to heat stress. Plant Responses to Abiotic Stress 4: 73-101
Larkindale J, Hall JD, Knight MR, Vierling E (2005) Heat stress phenotypes of arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiology 138: 882-897
Larkindale J, Vierling E (2008) Core genome responses involved in acclimation to high temperature. Plant Physiology 146: 748-761
Lee DH, Goldberg AL (1998) Proteasome inhibitors cause induction of heat shock proteins and trehalose, which together confer thermotolerance in Saccharomyces cerevisiae. Molecular and Cellular Biology 18: 30-38
Lee GJ, Vierling E (2000) A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein. Plant Physiology 122: 189-197
Lee JH, Schoffl F (1996) An Hsp70 antisense gene affects the expression of HSP70/HSC70, the regulation of HSF, and the acquisition of thermotolerance in transgenic Arabidopsis thaliana. Molecular & General Genetics 252: 11-19
Lee U, Wie C, Escobar M, Williams B, Hong SW, Vierling E (2005) Genetic analysis reveals domain interactions of Arabidopsis Hsp100/ClpB and cooperation with the small heat shock protein chaperone system. Plant Cell 17: 559-571
Lin BL, Wang JS, Liu HC, Chen RW, Meyer Y, Barakat A, Delseny M (2001) Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana. Cell Stress & Chaperones 6: 201-208
Liu XQ, Xu LF, Liu YW, Tong XH, Zhu GY, Zhang XJC, Li XM, Rao ZH (2009) Crystal structure of the hexamer of human heat shock factor binding protein 1. Proteins-Structure Function and Bioinformatics 75: 1-11
Lohmann C, Eggers-Schumacher G, Wunderlich M, Schoffl F (2004) Two different heat shock transcription factors regulate immediate early expression of stress genes in Arabidopsis. Molecular Genetics and Genomics 271: 11-21
Miller G, Mittler R (2006) Could heat shock transcription factors function as hydrogen peroxide sensors in plants? Annals of Botany 98: 279-288
Morimoto RI (1998) Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes & Development 12: 3788-3796
Morrow G, Inaguma Y, Kato K, Tanguay RM (2000) The small heat shock protein Hsp22 of Drosophila melanogaster is a mitochondrial protein displaying oligomeric organization. Journal of Biological Chemistry 275: 31204-31210
Never L, Scharf KD, Gagliardi D, Vergne P, CzarneckaVerner E, Gurley WB (1996) The Hsf world: Classification and properties of plant heat stress transcription factors. Cell Stress & Chaperones 1: 215-223
Nishizawa A, Yabuta Y, Yoshida E, Maruta T, Yoshimura K, Shigeoka S (2006) Arabidopsis heat shock transcription factor A2 as a key regulator in response to several types of environmental stress. Plant Journal 48: 535-547
Nover L, Bharti K, Doring P, Mishra SK, Ganguli A, Scharf KD (2001) Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need? Cell Stress & Chaperones 6: 177-189
Paine PL, Moore LC, Horowitz SB (1975) NUCLEAR-ENVELOPE PERMEABILITY. Nature 254: 109-114
Panikulangara TJ, Eggers-Schumacher G, Wunderlich M, Stransky H, Schoffl F (2004) Galactinol synthase1. A novel heat shock factor target gene responsible for heat-induced synthesis of raffinose family oligosaccharides in arabidopsis. Plant Physiology 136: 3148-3158
Pirkkala L, Nykanen P, Sistonen L (2001) Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. Faseb Journal 15: 1118-1131
Queitsch C, Hong SW, Vierling E, Lindquist S (2000) Heat shock protein 101 plays a crucial role in thermotolerance in arabidopsis. Plant Cell 12: 479-492
Sakuma Y, Maruyama K, Qin F, Osakabe Y, Shinozaki K, Yamaguchi-Shinozaki K (2006) Dual function of an Arabidopsis transcription factor DREB2A in water-stress-responsive and heat-stress-responsive gene expression. Proceedings of the National Academy of Sciences of the United States of America 103: 18822-18827
Satyal SH, Chen DY, Fox SG, Kramer JM, Morimoto RI (1998) Negative regulation of the heat shock transcriptional response by HSBP1. Genes & Development 12: 1962-1974
Schramm F, Ganguli A, Kiehlmann E, Englich G, Walch D, von Koskull-Doring P (2006) The heat stress transcription factor HsfA2 serves as a regulatory amplifier of a subset of genes in the heat stress response in Arabidopsis. Plant Molecular Biology 60: 759-772
Shivkamat P, Roy R (2005) Regulation of membrane lipid bilayer structure during salinity adaptation: A study with the gill epithelial cell membranes of Oreochromis niloticus. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology 142: 28-36
Tai LJ, McFall SM, Huang K, Demeler B, Fox SG, Brubaker K, Radhakrishnan I, Morimoto RI (2002) Structure-function analysis of the heat shock factor-binding protein reveals a protein composed solely of a highly conserved and dynamic coiled-coil trimerization domain. Journal of Biological Chemistry 277: 735-745
Vierling E (1991) The roles of heat-shock proteins in plants. Annual Review of Plant Physiology and Plant Molecular Biology 42: 579-620