阿拉伯芥HD2型組蛋白去乙醯化酵素於非生物性逆境反應中的功能

Function of HD2 Histone Deacetylases in Abiotic Stress Responses in Arabidopsis

王囿媛(Yu-Yuan Wang)

指導教授 ： 吳克強

國立臺灣大學/生命科學院/植物科學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00123

摘要

真核生物染色質裡，DNA纏繞組蛋白形成核小體，而組蛋白的修飾是個調控基因表現很重要的機制。催化從組蛋白移除乙醯基的酵素稱為組蛋白去乙醯化酵素(histone deacetylase, HDAC)。植物組蛋白去乙醯化酵素有四個基因家族，前三個家族RPD3，HD1和SIR2在酵母菌及哺乳動物中都可以發現同源基因；第四個家族HD2則是植物特有的。阿拉伯芥中目前發現兩個SIR2蛋白(AtSRT1和AtSRT2)，以及四個HD2蛋白(AtHD2A, AtHD2B, AtHD2C和AtHD2D)，為了要了解SIR2和HD2兩群組蛋白去乙醯化酵素在植物對抗非生物性逆境中的功能，本研究中分析得到和失去功能(gain/loss-of-function)的兩種突變體在非生物性逆境的反應。
研究發現，SIR2和HD2的基因表現都會被離層酸(ABA)和鹽處理所抑制。過量表現HD2C基因的植株相較於野生型植株在種子發芽時期對離層酸和鹽逆境都較不敏感，幼苗在土壤中也對鹽逆境有較高的耐受性。HD2C的T-DNA插入剔除(knock-out)株hd2c-1則顯示相反的結果，在種子發芽時期對離層酸和鹽逆境高度敏感，幼苗在土壤中對鹽逆境耐受性較低。經過離層酸處理後，過量表現HD2C基因的植株會誘導較多對離層酸反應的基因(ABI1, ABI2和RD29B)，而在鹽處理後也同樣誘導較多對鹽反應的基因(DREB2A, RD29A, ABF4和RD29B)。利用染色質免疫沉澱(Chromatin immunoprcipitaiton)的方法分析ABA及鹽反應基因，結果顯示這些離層酸和鹽反應基因的組蛋白三乙醯化(H3ac)程度和組蛋白三上第四號賴胺酸三甲基化(H3K4me3)程度都會因為離層酸以及高鹽處理而提高，但其程度在野生型和突變株中並無顯著的差異。根據這結果推測可能HD2C並不直接調控這些基因的組蛋白修飾，但在植物對抗離層酸及鹽逆境反應中仍佔有重要的角色。相較於HD2C突變株，SIR2的過量表現突變株和T-DNA剔除株在離層酸及高鹽逆境下的反應都與野生型植株沒有什麼顯著的差異，顯示SIR2可能並不參與在這兩種反應的途徑中。利用雙分子螢光複合物分析(BiFC)研究結果顯示，HD2蛋白(HD2A，HD2C和HD2D)和RPD3群蛋白中的HDA6和HDA19能有交互作用，這表示HD2蛋白可能藉由與RPD3蛋白構成一個複合體來調控基因表現。

關鍵字

阿拉伯芥 ； 組蛋白 ； 去乙醯化酵素 ； 離層酸 ； 非生物逆境

並列摘要

In eukaryotic chromatin, DNA wind around the core histone proteins to form nucleosomes. Modification of histones is an important mechanism to regulate gene activity. The enzymes that catalyze removal of acetyl groups from the core histones are called histone deacetylases (HDACs). Plant HDACs can be grouped into four families. RPD3, HDA1, and SIR2 are homologous to the HDACs found in yeast and animal cells. The forth family of histone deacetylases, HD2, is plant-specific. Two SIR2 proteins, AtSRT1 and AtSRT2, as well as four HD2 proteins, AtHD2A, AtHD2B, AtHD2C and AtHD2D, have been identified in Arabidopsis. In this study, gain-of-function and loss-of-function mutants were analyzed to reveal the function of SIR2- and HD2-type histone deacetylases in abiotic stresses.
It is found that SIR2 and HD2 gene expression was repressed by ABA and NaCl treatment. Compared to wild-type plant, HD2C overexpression line showed lower sensitivity to ABA and NaCl during seed germination stage, and showed more tolerance to salt stress in seedling stage when grown in soil. hd2c-1, the T-DNA knock-out line of HD2C, was shown to have increased sensitivity to ABA and NaCl during germination, and decreased tolerance to salt stress. After ABA treatment, HD2C overexpression plants displayed increased expression of ABA-responsive genes including ABI1, ABI2 and RD29B. In addition, HD2C overexpression line also displayed increased expression of salt-responsive genes, DREB2A, RD29A, ABF4 and RD29B. The H3 acetylation and H3K4 trimethylation level of these ABA- and salt-responsive genes was analyzed by chromatin immunoprecipitation assay. Both H3 acetylation and H3K4 trimethylation level of the ABA- and salt- responsive genes could be induced by ABA and salt stress treatment, but there is no significant difference between wild-type and HD2C mutants. These data suggested that HD2C might not directly regulate the histone modification of these genes, but still plays an important role in plant ABA and salt responsive pathways. Unlike HD2C mutants, both SIR2 gain-of-function and loss-of-function mutants did not display significant difference compared to wild-type in the ABA and salt response, suggesting that SIR2 may not be involved in ABA and salt response pathway. BiFC assay indicated that HD2 proteins, HD2A, HD2C and HD2D, interact with RPD3-type histone deacetylase, HDA6 and HDA19, suggesting that HD2 proteins may form a complex with RPD3-type HDACs to regulate gene expression.

並列關鍵字

Arabidopsis ； histone deacetylase ； ABA and abiotic stress

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