摘要:當生物體處於高溫逆境/熱休克(HS)時,會誘導熱休克蛋白質(HSP)的生成,避免細胞受到熱逆境的傷害,稱之為熱休克反應(HSR),此反應受到熱休克轉錄因子(HSF)的嚴格調控,為所有生物體共通的現象。阿拉伯芥熱休克轉錄因子結蛋白 (AtHSBP)表現於各個組織並受HS誘導,缺陷後導致種子的發育不良,暗示可調控胚胎發育。AtHSBP表現於細胞質,細胞在由HS回復到正常情況下,會由細胞質轉移到細胞核內;長時間回溫後的細胞核偵測不到AtHSBP的表現。AtHSBP除了自身外也可與HSF進行交互作用,降低HSF與HSE結合的能力,降低HSF的轉錄活性。AtHSBP缺陷突變株,HSP基因表現量及HSP蛋白累積量比野生型多,具較高的耐熱性;在AtHSBP的過量表現株中則相反,導致耐熱性下降。這些新發現對於植物的熱逆境調控機制提供新的視野,並揭露AtHSBP在HSR扮演一個負向調控者並且與對於種子發育及作物產量具重要性。本計劃將更深入探討其AtHSBP之生理功能。除此、HSP對於植物耐熱性扮演重要的角色,但HS理後於回温過程當中處理Ca2+螯合劑EGTA,影嚮HSP的合成及累積也不影嚮其提供熱保護能力,但影嚮細胞膜的通透性,促使胞內液滲漏更加嚴重,剝奪植物HSP提供誘導耐熱性的能力。若添加二價陽離子(特別是鈣離子)即可恢復對此溫度的耐受性。我們證實HS能提高細胞壁果膠甲基酯酶(pectin methylesterase,PME)的活性,誘發細胞壁結構性Ca2+的移動,增加細胞質Ca2+ oscillation之訊號強度及頻率,並調控細胞壁中膠層Ca2+-pectate複合體的重組,參與回溫過程中植物細胞壁之重建,以增強細胞壁結構及維持細胞膜的完整性與植物抵抗熱逆境能力關。我們共篩選到二株阿拉伯芥PME的T-DNA插入突變株,均顯示耐熱性下降。本研究將會利用遺傳學證明PME對於植物耐熱性獲得的重要性。此外、植物對於外界各種刺激所產生之特定生理反應過程中,Ca2+扮演胞內信使的作用。藉由Ca2+螢光指示劑偵測水稻細胞質內Ca2+濃度的變化,我們證實HS能促進胞外的Ca2+之移動,藉由細胞膜Ca2+通道進入細胞,增強細胞質Ca2+ oscillation的訊號強度及頻率,此訊號可由特定的鈣調素(calmodium, CaM; OsCaM1-1)接收並與Ca2+結合後活化,再將信息傳遞至下游基因,影響HSP基因的表現。在阿拉伯芥中大量表現水稻OsCaM1-1基因,在常溫中即可增加HSF和HSP基因的表現及HSP蛋白質的累積,提高阿拉伯芥的耐熱性。我們建立了Ca2+及特定的OsCaM1-1參與水稻的耐熱性。本研究將更深入探討OsCaM1-1下游分子,建立在水稻中完整的HS訊息傳遞鍵。我們的研究工作促進吾人對於植物高溫逆境反應的認識,並提供改良耐熱性的可能線索。
Abstract: Heat shock response (HSR) is a universal mechanism in all organisms. It is under tight regulation by heat shock factor (HSF) and heat shock protein (HSP) after heat shock (HS) to prevent stress damage. Investigation of the role of HSF binding protein (HSBP) in Arabidopsis thaliana showed the cytosolic-localized AtHSBP translocates to the nucleus during the recovery from HS, a pattern that differed from that of human HsHSBP1, a predominantly nuclear-localized protein unaffected by HS. AtHSBP interacts with HSF negatively affect the DNA-binding capacity plays as a negative regulator of HSR and involves in control of flowing time, also crucial for seed development. HS during the reproductive growth period causes a serious grain yield loss and small HSP synthesis affects the quality in rice. In this project, we would like to pursue further in elucidating the molecular mechanism underlie the physiological function of AtHSBP particularly in control of flowering time and seed development. Using crops of rice and soybean, we showed HS leads to Ca2+ releases from the apoplast to cytosol in a typical “Ca2+ signature”, confers cell wall rigidity and enhances HS signaling pathway. We identified a cell wall protein, pectin methylesterase (PME), as requires during HSR for cell wall remodeling and plasma membrane integrity. PME is activated in response to HS and its elevated activity causes an increased level of demethylesterified pectin, which is related to the recovery of HS-released Ca2+ concentration. Thus, the recovery of HS-released Ca2+ in Ca2+-pectate reconstitution through PME activity is required for cell wall remodeling during HS, which in turn retains plasma membrane integrity and coordinates with HSP to confer thermotolerance. This finding confirmed in addition to HSP, other cell-wall factors are involved for thermotolerance establishment. In our study, a screening of 53 Arabidopsis PME-knockout lines revealed two mutants consistent reduction of thermotolerance. This finding supported that PME has a novel role in thermotolerance. Here, we will address the specific PME gene is essential for acquisition of thermotolerant genetically. We confirmed HS-induced the entry of apoplastic Ca2+ is responsible for the biphasic calcium ([Ca2+]cyt) signature transduces by a Ca2+ sensor protein, the calmodulin OsCaM1-1, which triggers the downstream HS-responsive components leading to acquired thermotolerance in rice. Nuclear localization of OsCaM1-1-GFP follows the biphasic [Ca2+]cyt signature, which may provide the necessary information to direct the expression of HS-responsive genes during HSR. Moreover, overexpression of rice OsCaM1-1 in Arabidopsis effectively modulated the intensity of the HSR and enhanced thermotolerance. Thus, OsCaM1-1 plays as a coordinator of HSR to decode the Ca2+ signature to transmit HS signal. We would also like to identify the OsCaM1-1-mediated downstream effectors, including protein kinases and transcription factors, in order to decipher the complexity of HS signaling pathway in rice. In summary, our studies on the major stress affecting plant development and productivity, HS, focus on the new key components in HS pathway could effectively improve our understating the complexity of HS signaling and bring possible clues for improvement of HS tolerance.