阿拉伯芥中的SCE1和COP1會透過泛素化與類小泛素化調節轉錄因子ERF1在光照與黑暗下的蛋白質穩定性

Abstract

ETHYLENE RESPONSE FACTOR 1 (ERF1)是阿拉伯芥當中參與在生物性與非生物性逆境中，一個極為重要的轉錄因子，並且主要調控乙烯訊息傳遞路徑。先前已有研究指出ERF1蛋白在黑暗中會受到UBIQUITIN-CONJUGATING ENZYME 18 (UBC18)的泛素化調控，經過酵素的合作，會負責標記目標蛋白使其透過proteasome的途徑降解而趨於不穩定。在本研究中，我們發現SUMO-CONJUGATING ENZYME 1 (SCE1)與ERF1有交互作用。同時另一個在阿拉伯芥當中扮演E3 ubiquitin ligase的蛋白CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1)，在黑暗下會藉由調控ERF1的ubiquitination而使其降解，反之在光照下SCE1會透過SUMOylation使ERF1蛋白趨於穩定。我們更進一步發現，當ERF1蛋白的SUMOylation位點被突變 (ERF14KR) 後，ubiquitination和SUMOylation的修飾都有減少的現象，並且在植物當中我們也觀察到在進入黑暗後ERF14KR沒有被降解，間接證實了對於ERF1，ubiquitination和SUMOylation存在著光照條件之間的競爭關係。不僅如此，我們也看到sce1突變株不管在鹽處理或是乾旱下，側根數量以及植株存活率都較WT來的低。同時我們也發現sce1突變株在黑暗處理下，ERF1的下游基因P5CS1以及OSM34的表現量都較WT更低，表示SCE1可以藉由調控ERF1的穩定性正向地參與在植物逆境反應中。綜上所述，我們的研究證實了一項新的調控ERF1蛋白穩定性的分子機制，以及SCE1在逆境訊息傳遞當中扮演的重要角色。

ETHYLENE RESPONSE FACTOR 1 (ERF1) is an important transcription factor which involves in biotic and abiotic stress, and plays a major role in ethylene signaling. Previous studies have shown that ERF1 is unstable in the dark and its degradation is mediated by UBIQUITIN-CONJUGATING ENZYME 18. Here, we demonstrated that SUMO-CONJUGATING ENZYME 1 (SCE1) can physically interact with ERF1 in plants. CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) is an E3 ubiquitin ligase that target substrate protein for proteosome degradation pathway. In vitro and in vivo ubiquitination and SUMOylation assays suggest that COP1 mediates ERF1 ubiquitination in the dark while SCE1 mediates ERF1 SUMOylation in the light. Moreover, in vitro ubiquitination assay showed that the SUMOylation sites-mutated ERF1 (ERF1-4KR) led to less ubiquitination compared to wild-type ERF1, suggesting that ubiquitination of ERF1 might compete with its SUMOylation on the same residues. Our drought- and salt-stress analyses also suggest that SCE1 plays a positive role in stress response. sce1 mutants showed less tolerant phenotype under both drought and high salinity. The induction of ERF1’s downstream genes such as P5CS1 and OSM34 are negatively regulated by SCE1 under light/dark cycle. Collectively, this study reveals the molecular mechanism regulating ERF1’s stability and light-stress signaling crosstalk.