阿拉伯芥WRKY63參與開花時間調控之分子功能研究

Abstract

植物的開花是生命週期中的一大重要環節，而開花時間的變化會藉由許多基因激活因子和抑制因子組成的複雜網絡進行調控。在阿拉伯芥（Arabidopsis thaliana）中，FLOWERING LOCUS C (FLC) 作為一個關鍵調控因子，透過抑制開花整合因子 FLOWERING LOCUS T (FT) 的表達，參與植物開花的調控。長時間的低溫處理（春化作用）能誘導非編碼長 RNA（lncRNAs）COOLAIR 和 COLDAIR 的表達量上升，間接抑制 FLC 表達來促使植物在春化條件下開花。然而，春化作用影響COOLAIR 和 COLDAIR 的轉錄調控機制尚不清楚。在本研究中，我們發現轉錄因子 WRKY63藉由活化FLC 的方式來調控植物的開花。在開花表型的研究中，wrky63突變體表現出早花的表型，且對春化作用不敏感，說明WRKY63的功能在非春化及春化作用中對植物開花的調控都扮演重要角色。在非春化條件下，WRKY63 可直接結合FLC啟動子區域，激活 FLC 的表達；然而在春化作用的影響下，WRKY63通過直接結合在COOLAIR 和 COLDAIR啟動子區域的方式，促進 COOLAIR 和 COLDAIR 的轉錄，而間接抑制 FLC的表達。全基因體結合分析顯示，春化作用會促進WRKY63 對春化誘導基因的結合程度，同時也伴隨抑制性組蛋白標誌 H3K27me3 水平的降低。綜上所述，這些研究結果證明 WRKY63是開花的關鍵調控因子，參與了春化作用影響下的基因轉錄調控。 蛋白質的轉譯後修飾（PTMs）在真核細胞中調節蛋白功能方面發揮著重要作用。在這些修飾中，蛋白質乙醯化作為一種進化上保守的 PTM，影響蛋白質的細胞位置、穩定性、蛋白質間相互作用、DNA 結合能力和酵素活性。近期的研究發現WRKY蛋白的功能可通過乙醯化進行調節，然而其分子機制並不清楚。在本研究中，我們鑑定出WRKY63的賴胺酸 31（K31）作為一個關鍵乙醯化位點，可以促進WRKY63的蛋白位置的改變及其活性與功能的調節。此外，WRKY63的乙醯化狀態改變，可以藉由組蛋白乙醯轉移酶 HAG1 (GCN5) 和組蛋白去乙醯酶 HDA6 的拮抗調節。我們的研究揭示了一種通過乙醯化調節 WRKY 蛋白功能的新機制。

Flowering time is a vital developmental process in plants, orchestrated by a complex network of gene activation and repression mechanisms. In Arabidopsis thaliana, FLOWERING LOCUS C (FLC) serves as a key regulator by repressing the expression of the floral integrator FLOWERING LOCUS T (FT). Prolonged cold exposure (vernalization) promotes flowering by downregulating FLC expression. The long noncoding RNAs (lncRNAs) COOLAIR and COLDAIR, transcribed from the 3' end and the first intron of FLC, respectively, play crucial roles in FLC repression during vernalization. However, the transcriptional activation mechanisms of COOLAIR and COLDAIR remain unclear. In this study, we identified the group-III WRKY transcription factor WRKY63 as a direct activator of FLC. wrky63 mutants exhibit an early flowering phenotype and reduced sensitivity to vernalization. WRKY63 directly binds to the promoters of COOLAIR and COLDAIR, activating their expression. Under non-vernalization conditions, WRKY63 directly activates the expression of FLC. During vernalization, it indirectly represses FLC by promoting COOLAIR and COLDAIR transcription. Genome-wide occupancy analysis revealed that WRKY63 binding to vernalization-induced genes increases following vernalization. Furthermore, WRKY63 binding correlates with reduced levels of the repressive histone marker H3K27me3. Collectively, these findings establish WRKY63 as a pivotal regulator of flowering, mediating transcriptional responses to vernalization. Post-translational modifications (PTMs) play a critical role in modulating protein functions in eukaryotic cells. Among these, protein acetylation is an evolutionarily conserved PTM that influences various aspects of protein, including subcellular localization, stability, protein-protein interactions, DNA-binding capacity, and enzymatic activity. The members of the WRKY protein family have recently been shown to undergo functional and activity regulation through acetylation. However, the specific effects of WRKY acetylation on their functions and activities are still unclear. In this study, we identified lysine 31 (K31) as a key acetylation site on WRKY63 that enhances its nuclear localization and transcriptional activity. Furthermore, the acetylation status of WRKY63 is antagonistically regulated by the histone acetyltransferase HAG1 (GCN5) and the histone deacetylase HDA6. Together, our findings reveal a novel mechanism by which acetylation modulates WRKY protein function.