HDA15互作蛋白之鑑定與分析

Abstract

組蛋白去乙醯酶(HDACs或者HDAs)主要作用於組蛋白N端離氨酸的去乙醯化作用，而此修飾在植物生長發育過程中扮演重要角色。組蛋白去乙醯酶HDA15為RPD3/HDA1 superfamily的成員，目前已知的功能為與PIF3共同调控葉綠素的生合成與光合作用。我們使用LC-MS/MS找到了許多可能與HDA15有交互作用的蛋白，並進一步以雙螢光分子雜交技術（BiFC）來確認這些蛋白與HDA15的交互作用。其中，我們發現HDA15與會參與植物抗病反應的MOS4 Associated Complex有交互作用。HDA15 突變株接種Pseudomonas syringae pv. tomato DC3000 3天後表現出對病原菌敏感，而此一病徵與mos4-1 的表型相似，說明HDA15可能參與在植物抗病反應中調控植物的免疫反應。 BIM1，BIM2和BIM3為bHLH的成員，會與BES1共同作用在E-box的啟動子區域上去調控Brassinosteroid反應基因。依據先前文獻，bim1/bim2/bim3三重突變會造成BR調控基因的反應下降。在本研究中，我們發現BIM2會與HDA15以及HDA6有交互作用。此外，HDA6與突變株bim1/bim2/bim3三重突變株在高鹽逆境下會出現花青素累積量下降，以及在種子萌發時期對高鹽逆境敏感的現象。

Histone deacetylases (HDACs or HDAs) are responsible for the deacetylation of lysine residues on the N-terminal tail of core histones and play an important role in transcriptional regulation, cell cycle progression and developmental events. HISTONE DEACETYLASES 15 (HDA15), one of the RPD3/HDA1 superfamily members, is known to regulate chlorophyll biosynthesis and photosynthesis by interacting with PHYTOCHROME INTERACTING FACTOR3 (PIF3). We found that many proteins can interact with HDA15 by LC-MS/MS analysis. The interaction of HDA15 with these identified proteins was further confirmed by bimolecular fluorescence complementation assays. In particular, we found that HDA15 can interact with the MOS4 associated complex involved in plant immune responses. Similar to mos4-1, hda15 mutants were hypersensitive to Pseudomonas syringae pv. tomato DC3000 after three days inoculation, supporting that HDA15 may interact with the MOS4 associated complex to regulate plant immunity. BES1-INTERACTING MYC-LIKE PROTEINS 1 (BIM1), BIM2 and BIM3 belong to the bHLH protein family. BIMs interact with BR-INSENSITIVE-EMS-SUPPRESSOR1 (BES1) to synergistically bind to the E-boxes that are present in the promoter regions of a multitude of genes such as Brassinosteroid (BR) responding genes. It was reported that the simultaneous elimination of all three BIM proteins results in reduced BR-mediated responses. In this study, we found that BIM2 can interact with HDA15 and HDA6, and bim1/bim2/bim3 and axe1-5 accumulate less anthocyanin under high salt conditions and are hypersensitive to salt stress in the seed germination stage.