阿拉伯芥鋅手指蛋白3在非生物逆境下的功能探討

Abstract

熱休克反應（HSR）是生物體的一種保守機制，它可透過熱休克因子（HSF）來大量誘導熱休克蛋白（HSP）的表達。這種機制可以使細胞在高溫逆境來臨的時候，可以擁有更高的存活機會，使得細胞可以在足以致命的高溫中存活。在之前的研究中，我們發現阿拉伯芥的熱休克因子HSFA6b可以透過離層酸（ABA）來調節植物在熱逆境中的反應，HSFA6b在ABA調節的耐熱性中扮演非常重要的角色。有趣的是，HSFA6b的表達量並不會受到高溫的誘導，因此我們提出了一個全新的ABA訊號傳遞的機制，這讓我們得以一窺以ABA為主調控的耐熱網絡。然而，這種ABA調控網絡如何與復雜的熱休克反應網絡交互作用仍然不清楚。在HSFA6b的進一步研究中，我們發現阿拉伯芥的Cys2 /His2型鋅手指蛋白3 （AZF3）轉錄因子會受到HSFA6b調控，且在熱逆境的處理下表達量有顯著上升，而且相較於熱處理，其他逆境處理下的表現量則提升有限。在微陣列晶片資料顯示中，AZF3的基因表達水平會受到熱的強烈影響，同時還會受到HSFA6b和HSFA1的調節，這表明AZF3會是連接熱休克反應和ABA訊號網絡的重要因子。所以在這裡，我試著去了解HSFA6b調控的熱休克反應所需要的轉錄因子AZF3。在azf3突變株中ABA和熱反應相關的基因表達在37℃熱處理下會受到影響。雖然在ABA處理的培養基中，萌芽率沒有顯著性的差異，但是在先天耐熱性的處理下可以發現azf3突變株的存活率顯著性的高於WT。為了更進一步去研究這個現象，我建立了大量表現轉殖株AZF3-OE和剔除擁有潛在轉錄抑制功能的EAR motif的轉殖株AZF3-ΔEAR，同時建立了把EAR motif替換成VP16(轉錄活化motif)以及SRDX(轉錄抑制motif)的轉殖株。與azf3突變株相比，AZF3-ΔEAR和AZF3-VP16擁有顯著耐熱的外表型，相對而言AZF3-OE和AZF3-SRDX則擁有顯著對熱敏感的外表型。本篇研究說明了AZF3的表達受到熱和ABA的正向調控，並降低了正常熱休克反應所需的下游正向或負向調節子的表達。

Heat shock response (HSR) is a conserved mechanism in organisms that induces the expression of heat shock protein (HSP) through heat shock factor (HSF) mediation. This mechanism allows cells to have higher tolerance in heat stress (HS). In previous study, we showed that the Arabidopsis HSFA6b, one of the members of class-A HSF, plays an important role in the abscisic acid (ABA)-mediated thermotolerance. Interestingly, the expression of HSFA6b was HS independent, a novel ABA-mediated thermotolerance pathway thus proposed. However, how this new ABA signaling pathway connects to the complex HSR network still remains unclear. Further study in HSFA6b-overxpresion (OE) and -SRDX lines, we discovered that Arabidopsis Zinc Finger Protein 3 (AZF3) was tightly regulated by HSFA6b and HSFA1. Compared to HS, the expression level of AZF3 in various abiotic stress was limited. Here, we focused on AZF3, a transcription factor, required for this HSFA6b-mediated HSR. The expression of HS-responsive and ABA-related genes were affected in azf3 plants in response to 37°C-HS treatment. azf3 showed significantly higher thermotolerance phenotype than that of the WT by basal thermotolerance test. Nevertheless, there was no significant difference of seeds germination rate between WT and azf3 in response to ABA treatment. We generated transgenic lines of AZF3-OE, and its potential repressor domain EAR deletion lines of AZF3-ΔEAR. As well as, the EAR-motif was swapped with the VP16 (strong activation) or SRDX (strong repression) domain. The AZF3-ΔEAR and AZF3-VP16 lines showed significantly enhanced thermotolerance phenotype compared to the azf3. Accordingly, AZF3-OE and AZF3-SRDX lines showed significantly HS-sensitive phenotype compared to the azf3. This study demonstrates that the expression of AZF3 was positively regulated by heat and ABA and to attenuate the downstream negative/positive regulators expression that required for proper HS response.