SCE1 和 UBC18 與 ERF1 交互作用並調控其蛋白質穩定 性

Abstract

為了抵禦環境多重的改變，植物發展出複雜的調控系統來適應逆境，阿拉伯芥中的 Ethylene Response Factor 1 （ERF1）為乙烯及茉莉酸訊息傳遞的下游成員，並且對於防禦病原菌和非生物性逆境的反應扮演著正向調控的角色，在我們實驗室先前的研究中發現， ERF1 會在黑暗中不穩定並且藉由 26S 蛋白質酶體路徑而降解，透過酵母菌雙雜合系統(Y2H)，我們挑選到 51 個蛋白質可能會和 ERF1 交互作用，其中我們對於 UBIQUITIN-CONJUGATING ENZYME 18 (UBC18)和SUMO-CONJUGATING ENZYME 1 (SCE1)感到高度興趣，它們分別參與在泛素化和 SUMO 化路徑中。利用 pull down assay 和雙分子螢光互補系統(BiFC)進一步證實 SCE1 和 UBC18 會與 ERF1 交互作用。當利用農桿菌感染同時表現 ERF1 和UBC18 於菸草葉子中，越多的 UBC18 表現時，ERF1 表現會減少，表示說 ERF1的降解需要 UBC18 的參與。並且觀察到在黑暗處理後，相對於 WT，ubc18 knockdown 突變株有較多的 ERF1 累積，相反地，UBC18 過度表現植株則 ERF1 較少，這也表示說 UBC18 可能會調控 ERF1 於黑暗中的降解，另外也發現到 sce1 knockdown 突變株展現出對逆境敏感的表型，而 ubc18 knockdown 突變株展現出對逆境耐受的表型。綜合以上，在黑暗時，UBC18 可能會與 ERF1 交互作用並藉由26S 蛋白質酶體路徑使其降解，相反地，在光照時，SCE1 可能會與 ERF1 交互作用並藉由 SUMO 化來增加其穩定性。

Plants have developed a sophisticated regulatory system to adapt to various environmental stresses. The Arabidopsis ETHYLENE RESPONSE FACTOR 1 (ERF1) is a downstream component of jasmonic acid (JA) and ethylene (ET) signaling to positively regulate pathogen defense and is also involved in abiotic stress response. We found that ERF1 protein became unstable and was degraded through the 26S proteasome pathway under darkness. A yeast two-hybrid (Y2H) screening has identified 51 proteins that could interact with ERF1. Among these candidates, we were mostly interested in UBIQUITIN-CONJUGATING ENZYME 18 (UBC18) and SUMO-CONJUGATING ENZYME 1 (SCE1) which participate in ubiquitylation and sumoylation, respectively. Pull down assays and bimolecular fluorescence complementation assays were carried out to further confirm the interactions between SCE1, UBC18 and ERF1. When ERF1 and UBC18 were coexpressed in tobacco leaves using agro- infiltration, the increment of UBC18 coincided with the decrease of ERF1, indicating that UBC18 is required for the degradation of ERF1. Furthermore, we found that more ERF1 protein accumulated in ubc18 knockdown mutants, whereas less ERF1 protein accumulated in UBC18 overexpressers compared with that of wild type. These results suggest that UBC18 may mediate ERF1 degradation under darkness. We also found that sce1 knockdown mutants showed stress-sensitive phenotype while ubc18 knockdown mutants displayed stress tolerant phenotype. Taken together, UBC18 may interact with ERF1 to promote its degradation through 26S proteasome pathway in the dark. By contrast, SCE1 may interact with ERF1 to increase its stability via sumoylation in the light.