阿拉伯芥轉錄因子WRKY63與組蛋白去乙醯基酶HDA6相互作用並調控開花時間

Abstract

WRKY轉錄因子為植物特有之一群蛋白質家族，其特色在於具有至少一個保守的WRKY結構域，WRKY轉錄因子可結合至目標基因啟動子之W-box上，進一步調控目標基因的表現。目前研究發現WRKY轉錄因子參與在生物逆境、非生物逆境以及植物生長發育之調控。本文主要研究阿拉伯芥轉錄因子WRKY63在開花時間之調控機制，以及其與組蛋白去乙醯基酶HDA6互作之生理意義。 我們實驗室利用酵母菌雙雜交篩選發現WRKY63可以與HDA6進行交互作用，並利用雙分子螢光互補實驗以及共免疫沉澱驗證了此交互作用。我們的研究發現WRKY63可能藉由促進FLC (FLOWERING LOCUS C) 開花抑制子之表現，進而影響開花。WRKY63突變株abo3無論是在長日照亦或是短日照條件下皆比野生型Col-0早開花；而HDA6突變株axe1-5則皆為晚開花；然而abo3 axe1-5雙突變體植株則呈現中間型之表現型。我們發現在abo3中，FLC之mRNA表現量比野生型Col-0低。此外，WRKY63在體外或體內皆可被乙醯基化修飾，而此修飾與否會進一步影響WRKY63之轉錄活性。利用在原生質體進行短暫表現檢測轉錄活性之方式，我們發現HDA6可以抑制WRKY63之轉錄活性。這些實驗結果指出WRKY63與HDA6之交互作用可能在開花時間之調控上扮演著相當重要的角色。

WRKY transcription factors are one plant-specific class of proteins containing at least one conserved WRKY domain. WRKYs regulate the expression of genes involved in plant responses to biotic/abiotic stresses and plant development by targeting to the W-box directly. Previously, it was found that histone deacetylase HDA6 can interact with WRKY63 in yeast two-hybrid assays. The interaction of HDA6 and WRKY63 was further confirmed by bimolecular fluorescence complementation assays and co-immunoprecipitation assays. In this study, we found that WRKY63 regulates flowering by regulating the expression of FLOWERING LOCUS C (FLC). The wrky63 loss-of-function mutant, abo3, showed early flowering under both long day (LD) and short day (SD) conditions. In addition, the transcript level of FLC was down-regulated in abo3 compared to Col-0 wild type. In contrast, the hda6 mutant, axe1-5, showed late flowering under both LD and SD. The abo3 axe1-5 double mutant displayed an intermediate flowering phenotype compared to the single mutants. Furthermore, WRKY63 can be acetylated both in vitro and in vivo, and this post-translational modification can influence its transcriptional activity. Using transient transcriptional activity assays in Arabidopsis protoplasts, we found that HDA6 inhibits the gene activation ability of WRKY63. Together, these results suggest that the interaction of WRKY63 and HDA6 play a critical role in controlling flowering time in Arabidopsis.