油菜固醇調控阿拉伯芥之磷酸化蛋白質體研究

Abstract

油菜固醇是一固醇類植物荷爾蒙，在植物內廣泛的調控生長和發育。油菜固醇的訊息傳遞路徑為細胞表面的激酶受器偵測油菜固醇的訊號後，經由一系列的蛋白質磷酸化與去磷酸化，進行細胞內的訊息傳遞和調控基因表現，進而影響植物的生長與發育。蛋白質磷酸化為一種後轉譯修飾，對細胞內的訊息傳遞和植物的荷爾蒙調節有舉足輕重的影響。目前對於油菜固醇的研究多關注於基因和轉錄體層次的表現，對於磷酸化蛋白質體的相關研究相對少見。本研究目的為探討油菜固醇在植物體內調控的磷酸化蛋白質體。利用一具有根部特性之阿拉伯芥細胞，PSB-D，經油菜固醇處理後，結合雙甲基定量法和二氧化鈦豐富法，並利用液相層析與串聯式質譜儀 (LC-MS/MS)鑑定連續時間點中的磷酸化胜肽及其變化量。在本研究中，共發現了1481條磷酸化胜肽對應519個蛋白質，其中在402個可定量磷酸化胜肽中有12條磷酸化胜肽被判定受到油菜固醇的調控，其包含了核醣體相關蛋白，激酶，磷酸酶等。這些蛋白參與了轉錄，轉譯和生理代謝相關機制，其中可能包含過去未被發現經油菜固醇調控的蛋白質。本研究提供了油菜固醇在植物體所調控的磷酸化蛋白質變化的資訊，並有助於了解植物荷爾蒙在蛋白質磷酸化所扮演的角色

Brassinosteroid (BR) is a plant hormone involved in almost every stage of plant development. BR response starts from receptor kinase activation, and relays BR signal from cell surface to the nucleus by phosphorylation-mediated signaling. To date, the signaling pathway of BR has been well established at the gene expression level. However, only little information of phosphoproteins in BR signaling can be available. To investigate the global phosphorylation-regulatory responses in BR signaling, a time-dependent phosphoproteomic study was performed in Arabidopsis cells. Dimethyl labeling and TiO2 enrichment combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to characterize the phosphoproteome of BR signaling process. Our results identified 519 phosphorylated proteins from 1481 phosphorylated peptides. We further focused on significant change in quantification phosphoprotein. Changed in 12 of 402 quantitated phosphopeptides were significant. These proteins containing ribosome associated proteins, kinases and phosphatases are known to be involved in transcription, translation regulation and metabolic processes. Several proteins that may be regulated by BR were not known previously. This study uncovered a wide-range of phosphorylated proteins in response to BR signaling. Our results not only provide useful information for the phosphoproteomic research in plant hormone, but also reveal a new picture of signal transduction on BR regulation.