阿拉伯芥新穎蛋白質HHP1之功能研究

Abstract

HHP1蛋白質被預測具有七個穿膜區塊，是adiponectin接受體 (AdipoRs) 以及膜上progestin接受體 (mPRs) 的同源蛋白質。經由定量即時聚合酶鏈鎖反應與HHP1啟動子控制表現之GUS 活性分析，HHP1基因會被ABA及滲透逆境所誘導表現。由GUS活性的組織染色實驗發現，HHP1基因在某些器官及組織中有大量表現情形，這些器官及組織包括：根、維管組織、氣孔、水孔 、附著區塊及隔膜與種子的連接處，此結果顯示在阿拉伯芥中，HHP1可能參與膨壓改變及外來滲透逆境反應。此外，相對於野生型 (WT)，在發芽率、早期生長與逆境反應基因 (RD29A、RD29B、ADH1、KIN1、COR15A及COR47) 表現的分析實驗中，HHP1的T-DNA插入基因剔除突變株 (hhp1-1) 對外加的ABA與滲透逆境具有較高敏感度。此種高敏感度在hhp1-1互補突變株 (c-hhp1-1) 中，可以被表現正常的HHP1基因所回復。然而，在保衛細胞中，相較於野生型與c-hhp1-1突變株，hhp1-1突變株對ABA及乾旱逆境則會呈現較低的敏感度。 此外，另一個HHP1的T-DNA插入基因剔除突變株 (hhp1-2)，在轉譯成功狀況下，可以表現出只包含HHP1 N端的蛋白質片段。hhp1-2突變株對於ABA與滲透逆境的敏感度與hhp1-1突變株不盡相同，表示在HHP1的生理作用與滲透逆境之訊息傳遞上，HHP1的N端片段可能扮演重要角色。 藉由一級結構預測、細胞定位實驗與拓撲學分析，HHP1是一種細胞膜蛋白質，其C端片段朝向細胞外而N端片段 (第1至96個胺酸) 朝向細胞質內。酵母菌雙雜合實驗與雙分子螢光互補分析 (BiFC) 結果顯示，HHP1的N端片段與ICE1 (inducer of CBF expression 1) 有蛋白質交互作用，且交互作用的位置發生在細胞膜上。而外加冷逆境的狀況下，冷逆境反應基因 (RD29A、KIN1、COR15A及 COR47) 及會受ICE1所調控的CBF3與MYB15基因，在hhp1-1突變株中會有較低的基因表現情形，由此推測HHP1可能參與由ICE1傳遞的冷逆境訊息傳遞路徑。 在本論文中，我們推測HHP1基因突變會造成植物對ABA與滲透逆境有較高的敏感度，顯示在阿拉伯芥的早期生長時期，對於ABA與滲透逆境的訊息傳遞路徑而言，HHP1可能扮演一種負向調控因子的角色。此外，HHP1可能會透過與ICE1的蛋白質交互作用，來作為滲透逆境與冷逆境訊息傳遞的一個新交叉點。

HHP1 (heptahelical transmembrane protein 1), a protein with a predicted seven transmembrane domain structure homologous to adiponectin receptors (AdipoRs) and membrane progestin receptors (mPRs), has been characterized. Expression of HHP1 was increased in response to abscisic acid (ABA) and salt/osmotic stress as shown by quantitative real-time PCR and HHP1 promoter-controlled GUS activity. By histochemical analyses of GUS activity, the expression of HHP1 was predominantly shown in the roots, vasculature, stomata, hydathodes, adhesion zones, and the connection sites between septa and seeds in the HHP1::GUS transgenic mutants, indicating that HHP1 may participate in the turgor pressure changes and the responses to exogenous osmotic stress in Arabidopsis. In addition, the HHP1 T-DNA insertion knockout mutant (hhp1-1) showed a higher sensitivity to ABA and osmotic stress than the wild-type (WT), as revealed by the germination rate and post-germination growth rate. The induced expression of stress-responsive genes (RD29A, RD29B, ADH1, KIN1, COR15A, and COR47) was more sensitive to exogenous ABA and osmotic stress in the hhp1-1 than in the WT. The hypersensitivity in the hhp1-1 mutant was reversed in the complementation mutant of HHP1 (c-hhp1-1) expressing HHP1 gene. However, the guard cells in the hhp1-1 have an impaired response to drought and ABA stress compared with that in the WT and c-hhp1-1. Furthermore, the other HHP1 T-DNA insertion mutant, hhp1-2 (encoding a truncated N-terminus of HHP1), showed different sensitivity to ABA and osmotic stress compared with hhp1-1 by phenotypic observation and expression patterns of stress-responsive genes, implying that the N-terminus of HHP1 may play an important part in its function and mechanism on the osmotic signaling. Based on primary structure prediction, subcellular localization and topology analyses, HHP1 was a plasma membrane protein adopting a C-terminal-outside topology with an N-terminal region of about 96 amino acids facing the cytosol. By yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC), the N-terminal fragment (amino acids 1-96) of HHP1 was found to interact with ICE1 (inducer of CBF expression 1) while this interaction occurred in the plasma membrane of Arabidopsis protoplasts. The induced expressions of CBF3 and MYB15, which are regulated by ICE1, and cold stress-responsive genes (RD29A, KIN1, COR15A, and COR47) were less sensitive to exogenous cold stress in the hhp1-1 than in the WT, indicating that HHP1 may participate in the ICE1-mediated cold stress signaling pathway. In this study, we suggested that the mutation of HHP1 renders plants hypersensitive to ABA and osmotic stress, implying that HHP1 functions as a negative regulator in ABA and osmotic signaling during the early growth of Arabidopsis. Furthermore, HHP1 may act as a novel cross-talk point to the osmotic/cold signaling networks via ICE1.