阿拉伯芥E3 ligase RED1與RED2 在有性生殖與逆境反應之研究

Abstract

泛素化系統在許多生理功能具重要調控功能，而與目標蛋白高度專一結合的E3泛素接合酶 (E3 ligase)更扮演關鍵角色，然而目前大多數的E3 ligases尚未被研究。本研究室先前發現數個座落於微管且功能未知的植物RING-H2 type E3 ligases，將其命名為RING-type E3 ligase with an uncharacterized DAR1 domain (REDs)。本研究旨在了解阿拉伯芥RED1與RED2在有性生殖與逆境反應之功能。基因轉錄分析結果顯示RED1與RED2在花之表現量最高。酵母菌雙雜交分析結果顯示RED2形成homodimer需要DAR1 domain的存在，然而RED1可僅透過其RING domain與RED2形成heterodimer，且in vitro ubiquitination 結果顯示RED1與RED2可能會相互泛素化彼此。RED1與RED2之單基因缺失突變株並無生殖缺失，但其雙基因突變植物具明顯種子發育缺失，且雙基因突變植物與野生型植物之正反雜交試驗，結果顯示這兩個基因可能同時參與雌配子體與雄配子體之發育，雙基因突變植物之花粉管萌發率下降且胚株發育延遲。另外，利用阿拉伯芥原生質體搭配螢光觀察結果顯示RED1與RED2可能會影響葉部的微管動態。此外，RED1與RED2在乾旱與鹽害逆境反應可能未扮演重要的角色。以上結果指出RED1與RED2在阿拉伯芥有性生殖扮演關鍵之正向調控角色。為釐清其具體調控機制，目前正在製備各式互補回復株與將用於觀察微管動態之植物材料。我們期許目前所得之研究資訊與未來更進一步之研究有助於對E3 ligases有更完整的了解。

Protein ubiquitination is an important mechanism in regulating key cellular functions. The E3 ligases play critical roles in specific recognition of the targets; however, most plant E3 ligases are not characterized. We previously identified a group of uncharacterized plant microtubule-localized RING-H2 type E3 ligases, named RING-type E3 ligases with an uncharacterized DAR1 domain (REDs). This study aims to characterize roles of REDs in Arabidopsis reproduction and stress responses. Results of spatiotemporal transcriptional analyses by RT-qPCR assay showed that RED1 and RED2 highly express in flower. Yeast-two-hybrid (Y2H) assay revealed that the DAR1 domain is required for RED2 homodimerization, but not for RED1-RED2 interaction, and that RED1 and RED2 can form heterodimers via the RING domain of RED1. In addition, in vitro ubiquitination assay showed that RED1 and RED2 could ubiquitinate each other. red1red2 mutant plants, but not the single-gene mutants, displayed significant defects in seed production. Reciprocal cross assay revealed a critical role of RED1 and RED2 in both male and female gametogenesis, and the double mutants displayed defective pollen germination and ovule development. Microscope fluoresce assay showed that the properties of the microtubule in leaf might be affected in red1red2. Moreover, RED1 and RED2 may not play obvious roles in responses to drought and salinity stress. These results suggest that RED1 and RED2 play a vital positive role in Arabidopsis reproduction. To further elucidate the involved regulatory mechanisms, plant materials to be used for genetic complementation and microtubule dynamics analyses have being prepared. These results and the on-going studies are expected to help elucidate the regulatory role of these REDs in plant sexual reproduction.