WRKY63與HDA15之交互作用及分子功能分析

Abstract

植物開花時機受到內部和外部因素的精密調控，FLOWERING LOCUS C (FLC) 在抑制植物開花扮演重要的作用，它可抑制FLOWERING LOCUS T (FT) 和SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) 等與開花相關的基因表現，抑制開花的發生。WRKY63可促進FLC基因的表現，在過量表現HDA15之阿拉伯芥植株中，FLC的表現量呈現上升趨勢，而質譜分析的初步結果顯示，HDA15與WRKY63可能位於同一複合物中。然而，HDA15和WRKY63之分子結合與功能以及它們如何精細調控FLC基因的表達仍不清楚。在本研究中，蛋白質交互作用實驗pull down assay及Quartz crystal microbalance (QCM) 確認WRKY63藉由DNA結合區 (DB) 與HDA15之去乙醯基催化結構區 (HD) 結合。接著以酵素動力學分析WRKY63對於HDA15之去乙醯基活性的影響，結果顯示，WRKY63與帶有鋅指之HDA15 (HDA15-ZFHD) 結合後會促使HDA15-ZFHD之活性顯著上升。經以fEMSA及QCM對於WRKY63與FLC基因啟動子區域之W box的互動進行分析，結果顯示WRKY63-DB與FLC W1以及FLC W2之親合力較與FLC W3的親和力強度高。以小角度散射方法SAXS結合蛋白質同源結構模擬WRKY63-DB-FLC W1複合體結構，展示了FLC W box中可能與WRKY63結合的關鍵DNA序列保守位點。進一步以QCM和fEMSA分析WRKY63、HDA15及FLC W box的互動。QCM結果顯示，HDA15無法與FLC W box-WRKY63-DB複合體形成三個分子之複合體，並且無法競爭複合體中WRKY63-DB的能力；fEMSA的結果則再次表明HDA15並不會干擾WRKY63-DB與FLC W box結合之能力。本研究深入探討了WRKY63、HDA15及FLC W box之間的功能交互作用關係。將可提供表觀遺傳調控分子HDA15與WRKY63轉錄因子在FLC基因於開花調控分子機制之關鍵訊息。

The timing of plant flowering is intricately regulated by internal and external factors. FLOWERING LOCUS C (FLC) crucially inhibits flowering by suppressing the expression of genes like FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). WRKY63 enhances FLC gene expression, with Arabidopsis overexpressing HDA15 showing an increased trend in FLC expression. Preliminary mass spectrometry indicates that HDA15 and WRKY63 may form a complex. However, the molecular binding, functions, and fine regulation of FLC gene expression by HDA15 and WRKY63 remain unclear. This study utilized pull-down assays and Quartz Crystal Microbalance (QCM), confirming that WRKY63 binds to HDA15 through its DNA binding domain (DB) and HDA15's deacetylase catalytic structure region (HD). Enzyme kinetics analysis revealed that WRKY63, upon binding to zinc finger-containing HDA15 (HDA15-ZFHD), significantly enhances HDA15-ZFHD's deacetylase activity. Further analysis using fEMSA and QCM on the interaction between WRKY63 and the FLC gene promoter's W box region showed higher affinity of WRKY63-DB for FLC W1 and FLC W2 compared to FLC W3. Small-Angle X-ray Scattering (SAXS) with protein homology structure simulations illustrated the structure of the WRKY63-DB-FLC W1 complex, revealing the conserved DNA binding sites within the WRKY63-DB-FLC W1 complex. QCM and fEMSA analyses investigated WRKY63, HDA15, and FLC W box interactions. HDA15 fails to form a complex with FLC W box-WRKY63-DB and doesn't compete with FLC W box within it, as seen in QCM. fEMSA further verifies HDA15 doesn't disrupt WRKY63-DB binding to FLC W box. This study delves into WRKY63, HDA15, and FLC W box interactions, offering insights into HDA15 and WRKY63 interplay in FLC gene's W box regulation.