指導教授：靳宗洛臺灣大學：植物科學研究所牛中言Niu, Chung-YenChung-YenNiu2014-11-282018-07-062014-11-282018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/262837熱休克反應是一個對抗熱逆境的重要機制，其可以藉由熱休克轉錄因子的調控增加熱休克蛋白的表現。此外藉由植物逆境賀爾蒙離層酸 (ABA) 的訊息傳遞也會參與在後天耐熱性。分析微陣列晶片中的資料顯示，阿拉伯芥熱休克轉錄因子HSFA6b會被鹽、滲透壓和冷逆境誘導表現而不是在熱逆境。在本篇文章中我們確定了HSFA6b會參與在ABA訊息傳遞並且會提升熱耐受性。有趣的是，在先前的研究中顯示在ABA缺失及對ABA不敏感的突變體中，證實ABA相關的訊息傳遞對於HSFA6b的表現是必須的；藉此我們也利用在原生質體中的轉錄活性檢測，確定了HSFA6b啟動子中的ABRE (ABA反應序列) 對ABA和AREB1 (ABA反應結合蛋白1) 的感應是不可或缺的，除此之外DREB2A (乾旱反應序列結合蛋白2A)、HSP18.2 (熱休克蛋白18.2) 和APX2 (抗壞血酸過氧化物酶2) 也會受HSFA6b所調控。而利用HSFA6b的轉植株及其突變體進行實驗，我們也發現HSFA6b會正向調控參與在ABA媒介的鹽和乾旱的耐受性。在耐熱性測試的結果也顯示HSFA6b會參與在後天耐熱性的調控過程中。我們的研究結果說明了一條新的調控途徑，HSFA6b除了會參與在ABA媒介的逆境反應之外也會參與在熱逆境調控的複雜網路之中。The heat stress (HS) response (HSR) is a conserved mechanism developed to increase the expression of heat shock proteins (HSPs) via a heat shock factor (HSF)-dependent mechanism. As well, signaling by the stress phytohormone abscisic acid (ABA) is involved in acquired thermotolerance. Analysis of Arabidopsis (Arabidopsis thaliana) microarray databases revealed that the expression of HSFA6b, a class-A HSF, was increased with salinity, osmotic and cold but not HS. Here, we show that HSFA6b plays a pivotal role in the response to ABA and in thermotolerance in Arabidopsis. Intriguingly, in previous studies showed salt-inducible HSFA6b expression was downregulated in ABA-insensitive and -deficient mutants, and exogenous ABA application restored its expression in ABA-deficient plants, so the ABA signal is required for proper HSFA6b expression. Consequently, transcriptional activation assay of protoplasts showed that ABA treatment and coexpression of an ABA-signaling master effector, ABRE-binding protein 1 (AREB1), could activate the HSFA6b promoter. In addition, DREB2A, HSP18.2 and APX2 were regulated by heat shock factor A6b that enhanced their expression. Analysis of ABA responses in drought and salt tolerance in HSFA6b-null, -overexpression and -dominant–negative mutants indicated that HSFA6b is a positive regulator participating in ABA-mediated salt and drought resistance. Thermoprotection tests showed that HSFA6b was required for thermotolerance acquisition. Our study reveals a network in which HSFA6b operates as a downstream regulator of the ABA-mediated stress response and is required for HS resistance. This new ABA-signaling pathway is integrated into the complex HSR network in planta.摘要 I Abstract II Introduction 1 Heat Shock Response and Thermotolerance 1 Heat Shock Factors 2 Heat Shock Factors in Arabidopsis 2 ABA signaling and mediates thermotolerance 4 Oxidative stress gene APX2 5 Motivation and Objectives 6 Materials and Methods 7 Plant Materials and Growth Conditions 7 RNA Preparation, cDNA Synthesis and Real-Time Quantitative PCR 7 Generation of Transgenic HSFA6b Mutant Plants 7 Bimolecular Fluorescence Complementation (BiFC) assays 8 Protoplast Transactivation Assay 8 Thermotolerance, Superoxide Dismutase (SOD) Activity, H2O2 and Pigment Content Determination 8 Chromatin Immunoprecipitation (ChIP) Assay 9 Microarray Assay 9 Statistical analysis 10 Primers and Accession Numbers 10 Results 11 Arabidopsis Class-A HSF HSFA6b Is Induced by Salt, Osmotic and ABA Treatment, not by HS 11 ABA Treatment and AREB1/ABF2 Co-expression Enhanced HSFA6b Promoter Activity 11 HSFA6b-null, -OE and -dominant–negative Mutants Were Screened and Confirmed 12 Misregulated of ABA-biosynthesis and -responsive Gene Expression in Response to Salt and ABA Treatment in HSFA6b Mutants 12 HSFA6b Participated in Salt- and ABA-Mediated HSP Expression 13 HSFA6b and other HSFs can interact and form hetero- not homo-oligomers 14 HSFA6s double-knockout and HSFA3-null mutants were screened and confirmed 15 HSFA6b functions independently of HSFA6a and with HSFA3 for activation of HSP18.2 and APX2 promoter 15 HSFA6b Regulated Thermotolerance 15 HSFA6b Overexpression Conferred Drought and Salt Tolerance 16 Transcription Profiling of HSFA6b-OE and -RD transgenic plants 16 Discussion 18 HSFA6b, a Nuclear Factor, Responds to ABA Signaling via the ABRE/ABF-ABRE Regulon 18 HSFA6b, a Positive Regulator, Is Involved in ABA-mediated Salt and Osmotic Stress Responses 20 HSFA6b Mediated ABA-Induced Thermo-, Drought- and Salt-tolerance 20 Crosstalk of HSFA6 and DREB2A in HSR Gene Expression and Regulatory Networks 21 There are other HS- transcription factors may co-interaction with HSFA6b under HS 24 HSFA6b may mediated HSFA3 co-interaction required for thermotolerance under diverse stress conditions 24 Tables 25 Figures 32 References 49 Supplemental Data 58 Appendixes 654057340 bytesapplication/pdf論文公開時間：2016/08/25論文使用權限：同意有償授權(權利金給回饋本人)離層酸熱休克轉錄因子熱休克反應thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262837/1/ntu-103-R00b42023-1.pdf