Regulation of small heat shock protein genes expression by heat shock factors transcriptome in Arabidopsis

熱休克反應為生物體內普遍存在的逆境抵抗機制，會誘導數群分子量不同的熱休克蛋白質(heat shock protein, HSP)產生，以進行細胞保護。植物中低分子量熱休克蛋白質(small HSP, sHSP)的累積與種類是已知物種最多的一群HSP。關於高溫誘導表現的機制，已知會由一群熱休克因子(heat shock factor, HSF)來調控。阿拉伯芥與水稻中，分別具有包含A、B、C三個類群的21個和25個HSF基因成員，顯示在高等生物對於HSP表現的調控極為複雜。目前關於HSF調節sHSP基因在不同逆境下的表現機制仍然不清楚。本研究希望藉由不同逆境處理觀察HSF和sHSP表現間的關聯性，以初步推測個別的HSF作用並建立阿拉伯芥之熱休克蛋白調控體(HSP regulome)。此外，我們期望能建立一個負向篩選的突變株轉殖系統，以釣取更多有關調控sHSP訊息傳導路徑中，不同於HSF路徑上的因子。所以我們在數種化學誘導物處理下以RT-PCR同時檢測所有sHSP與HSF的表現，發現阿拉伯芥sHSP均受到高溫、胺基酸類似物以及砷處理而誘導表現，但是鎘處理則只誘導部分基因的表現，NO釋放物(sodium nitroprusside, SNP)則不影響sHSP基因的表現。然而部份HSF的表現並不會受所測試處理的影響；HsfA2和三個B class的HSF表現均受到高溫、胺基酸類似物及重金屬的誘導。HsfA7b誘導表現則是對高溫比對重金屬更為敏感。我們篩選出在HsfA6a有T-DNA插入的knock out突變株，而此HsfA6a缺失的突變株，在熱耐受性的獲得以及高溫下sHSP基因受誘導的情形皆不受影響。另外藉由Motifsampler軟體分析，一具保留性”GCGTTCA”的序列，存在於三個皆受到胺基酸類似物誘導的啟動子區域。Promoter::GUS 分析顯示這段區域對於誘導sHSP基因的表現有其重要性。本研究建立了阿拉伯芥中HSF和sHSP基因的表現圖譜，但是HSF作為sHSP調控機制上的作用還需要更多HSF knock out lines以及相關突變株的研究，這是未來仍要持續的工作。Heat shock (HS) response is a general physiological reaction to both prokaryotic and eukaryotic cells when temperature is suddenly elevated. The heat shock proteins (HSPs) induced by HS act as molecular chaperones to protect cells from HS damages. In plants, small HSPs (sHSP) represent the most abundant and complexes HSPs. And heat shock factors (HSFs) are known to involve in the regulatory mechanism of sHSP expression under HS stress. In Arabidopsis and rice, there are 21 and 25 HSFs, which are divided into three classes, respectively. That may implicate a complicated regulation mechanism (regulome) in higher plants. Other than heat shock, the regulation of sHSPs expression is still not clear. In this study, we studied the relationship between the sHSPs and HSFs expression under various stresses and to speculate the function of individual HSF. To investigate the details of the sHSPs regulome, we set up a negative selection system to screen mutants that can not respond to the HS-like induction. RT-PCR was used to analyze the HSF transcriptome to clarify the regulation of sHSP genes expression. We determined that all of sHSP genes were induced when seedlings were challenged with HS, amino acid analog (azetidine, Aze) and arsenite. Some of sHSPs, in response to cadmium and sodium nitroprusside (SNP, a NO donor), did not affect the expression of sHSPs. However, expression of some HSFs was not interfered by treatments. HsfA2a and three class B HSFs were induced by HS, Aze, and heavy metals treatments. The expression of HsfA7b was hyper inducted under HS than by other chemical inducers. One HsfA6a T-DNA knock-out line was identified. Thermoacquisition and HS-induction sHSP genes expression were not interrupted in the hsfA6a-KO line. Using the Motifsampler program to analyze three azetidine up-regulation sHSP-CI genes promoter, a non HSE with the conserved element, “GCGTTCA”, was identified. In promoter::GUS assay, confirmation of this element may play an important role in the HS response. In this study, we set up the expression profile of sHSPs and HSFs under HS and chemical inducers. But the individual HSF knock-out lines and other mutants are necessary to used to clarify the regulation of sHSPs expression by HSFs.Abstract in Chinese 3 Abstract in English 4 Abbreviations 6 Introduction Heat shock response and heat shock proteins 7 Small heat shock proteins in plants 7 Heat shock granules major consist of small heat shock proteins 8 Biological functions of small heat shock proteins 9 Expression of small heat shock proteins in response to non-heat stress 11 Regulation of small heat shock proteins 12 Basic structure of heat shock factors in plants 13 Heat shock factors in Arabidopsis 14 The signal transduction from stress to heat shock proteins 15 Materials and Methods Plant materials 18 Total RNA isolation and RT-PCR 18 Leaf PCR and T-DNA insertion knock-out lines isolation 19 Plasmid constructs 19 Plant transformation 20 GUS staining 21 Thermotolerance assay 21 Bioinformatics 22 Results Expression profiling of sHSPs in public microarrays 23 Expression profiling of sHSPs under stress 23 Expression profiling of HSFs under stress 24 The hsfA6a-KO plants shown no obvious defects in thermotolerance 25 Positive and negative selection system to screen factors other than HSFs 26 The conserved domain in the promoter of AtHsp17.4-CI, AtHsp17.6-CI, and AtHsp17.8-CI 27 Discussion Protein homeostasis is sufficient to arouse the expression of all sHSPs in Arabidopsis 29 The role of NO in the heat shock response 29 As and Cd induced sHSPs by different signaling pathways30 The regulatory mechanism of sHSPs expression shown a multisteps induction via HSFs 31 No obvious thermotolerance defect in hsfA6a knock-out line 32 Deletion of “GCGTTCA” motif slightly reduced gene expression under HS 33 Tables and Figures 34 References 49 Appendixes 5