利用前向式遺傳學從阿拉伯芥辨識出全新之植物耐熱基因及解析其耐熱機轉;Using a forward genetic approach for the identification of novel heat tolerance determinants and characterization of their protecting mechanisms in Arabidopsis

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Title:	利用前向式遺傳學從阿拉伯芥辨識出全新之植物耐熱基因及解析其耐熱機轉;Using a forward genetic approach for the identification of novel heat tolerance determinants and characterization of their protecting mechanisms in Arabidopsis
Authors:	王亮晴;(王亮晴):Lian-Chin Wang
Contributors:	生命科學研究所
Keywords:	耐熱基因;阿拉伯芥;heat tolerance;Arabidopsis
Date:	2012-04-10
Issue Date:	2012-06-15 20:11:34 (UTC+8)
Abstract:	植物為固著性的生物，無法自行選擇適合的生長環境，因此，植物演化出許多不同的機制以因應不利的生長環境。近年來因溫室效應導致全球氣溫上升，高溫逆境已嚴重影響農作物的品質及產量。了解植物如何抵抗高溫逆境，提高其對高溫的耐受能力，已是改良農作物的生長及產量的重要課題之一。為找出植物負責耐受高溫的遺傳決定因子，我們利用前向式遺傳研究法 (forward genetic approach) 篩選出對高溫不具耐受性之突變株，以基因體定位方式找出突變位址，並進一步研究造成此突變性狀之遺傳因子的功能及生理角色。 hit2即是應用此方法所篩選出的其中一棵突變株，其在正常生長環境下與野生型植株並無明顯差異，但hit2對於長時間高溫及短時間熱休克逆境均會失去耐受能力。此外，hit2對於methyl viologen所造成的氧化逆境及在高溫環境下光所誘發的氧化傷害均比野生型植株有較不耐受性的現象，由此得知，hit2也對氧化逆境過度敏感。經基因遺傳位址定位已知，突變基因為細胞核輸出接受器EXORTEIN1A，其功能為調節蛋白質在細胞核與細胞質之間的進出。由以上的結果顯示，在植物中有特定的細胞核輸出接受器幫助植物抵抗因高溫所誘發的氧化傷害，使其可以耐受高溫的逆境，而此細胞核輸出接受器因突變喪失功能時，並不會影響植物正常的生長與發育。 hit1-1為另一對高溫逆境不具耐受性之阿拉伯芥突變株。此外，hit1-1也會影響花粉管之生長。HIT1會與AtVps52p及AtVps54p有co-localization現象，且HIT1與AtVps52p及AtVps54p均會相互作用，形成類似Golgi associated retrograde protein的複合體，參與囊泡從endosome回至Golgi之間的運輸。實驗結果推測，hit1-1對於高溫的不耐受性，可能是由於細胞膜的完整性受損所導致，並非因高溫引發的過氧化物對細胞膜傷害所造成。此外，hit1-1對於高溫處理持續之時間較為敏感但對於溫度處理之強度影響較不顯著。綜合以上的結果推測，HIT1可能參與調節囊泡運輸進而幫助維持細胞膜的熱穩定性，而使植物可以耐受高溫逆境。Heat stress is a serious abiotic stress that affects plant survival and crop production. Plants have evolved diverse mechanisms to adapt to stressful conditions. Understanding the heat stress defense mechanisms of plants and enhancing their thermotolerance are critical issues for improving crop production and yield. To identify genetic determinants that are essential for plant heat tolerance, we used a forward genetic approach to isolate and characterize the heat-intolerant mutants. hit2 was isolated by growth inhibition under sustained high temperature conditions. The basal thermotolerance of hit2 to heat shock was also impaired. Furthermore, the growth and development of hit2 seedlings were more sensitive to inhibition with methyl viologen than those of wild-type seedlings, and the survival rate of hit2 seedlings in response to heat stress was affected by light exporsure, suggesting that hit2 is also hypersensitive to oxidative stress. The mutated locus was mapped to EXPORTIN 1A, which encodes a nuclear export receptor. These results demonstrated that a nuclear transport receptor that regulates nucleo-cytoplasmic trafficking is not necessary for normal growth and development, but this receptor is vital for plant heat tolerance, in part, by mediating the protection of plants against heat-induced oxidative stress. hit1-1 is another heat-intolerant mutant of Arabidopsis. HIT1 encodes a homolog of the yeast VPS53p protein that mediates the recognition between vesicles in retrograde trafficking and the late Golgi. HIT1 can interact with AtVPS52 and AtVPS54 to form the Golgi-associated retrograde protein complex. Mutations of HIT1 led to reduced pollen tube length and plasma membrane thermostability. Furthermore, hit1-1 was hypersensitive to long-term heat stress but not to sudden heat shock. This phenomenon correlates with the known effect of impaired membrane remodeling. These results imply that HIT1 participates in the vesicle trafficking required for pollen tube elongation and the thermal adaptation of the plasma membrane in response to long-term heat stress.
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