建立 GFP 氧化還原感應器用於測定水稻原生質體細胞質穀胱甘肽 的氧化還原變化

Abstract

細胞中的氧化還原變化會廣泛的影響植物的生長發育及逆境耐受性。植物細胞中普遍存在還原態的 GSH，GSH 和氧化態 GSH (GSSG) 的比率被視為判斷植物是否遭遇逆境的重要指標，因此維持細胞中 GSH 氧化還原的平衡對植物生長及耐逆境非常重要。過去分析 GSH 的方法往往是破壞整個組織或細胞後，再分析 GSH 的氧化還原變化，導致無法了解不同胞器內 GSH 氧化還原狀態。氧化還原偵測蛋白 roGFP (ratiometric Green Fluorescence Protein) 可利用於偵測不同胞器中氧化還原的變化，目前偵測的方法都是將 roGFP 穩定性轉殖於特定植物胞器，再分析植物器官中 GSH 隨氧化還原狀態而變化的螢光強度，因此需花費較多時間，為解決此問題，試驗中嘗試將一個對於 GSH 氧化還原更敏感的 GRX1-roGFP2，暫時性表現在水稻原生質體特定胞器中，希望能快速分析胞器內 GSH 的氧化還原變化。GRX1-roGFP2 融合蛋白會表達在細胞質，但是當 GRX1-roGFP2 的N端加上一段幫助蛋白質表達在粒線體和葉綠體的 Glutathione reductase 1 transit peptide，可使 GFP 螢光訊號表現在粒線體和葉綠體。轉殖 GRX1-roGFP2 到原生質體後，處理不同濃度的 DTT (還原劑) 或過氧化氫 (氧化劑)，會造成 GRX1-roGFP2 螢光強度有顯著的變化，反應出氧化與還原之狀態，顯示 GRX1-roGFP2 可應用於水稻原生質體系統，測定胞器中 GSH 氧化還原變化。為進一步測試此系統，試驗中將 GRX1-roGFP2 轉殖至 Glutathione reductase 2 (GR2)-RNAi 轉殖植物原生質體，GR2 為細胞質型 Glutathione reductase，轉殖 GRX1-roGFP2 後，共軛焦顯微鏡及盤式螢光儀偵測結果顯示細胞質 GSH/GSSG 比率顯著降低，顯示弱化水稻細胞質型 GR2 基因會增加細胞質氧化態 GSH，但降低還原態 GSH。這些結果顯示利用水稻原生質體來表達 roGFP 蛋白質是一個偵測 GSH 氧化還原簡單且快速的方法，可廣泛應用於植物胞器內氧化還原變化之偵測。

Cellular redox changes extensively impacted plant growth, development and stress tolerance. Glutathione (GSH) is an ubiquitous tripeptide thiol in the cells. The ratio of reduced GSH to oxidized GSH (GSSG) is an indicator of plant health. Therefore, maintaining cellular GSH redox homeostasis is important to plant growth, development and stress tolerance. The majority of glutathione redox research has been based on the chemical analysis of whole-cell extracts, which unavoidably destroy subcellular compartment-specific information. Recently, GFP based ratiometric redox-sensitive GFP (roGFP) protein was used to detect the redox status within different organelles by stable transformation. To generate a simple and rapid detection system, this study is aim to target a redox sensitive GRX1-roGFP2 to cytosol or plastids of rice protoplasts. A glutathione reductase 1 transit peptide was fused with GRX1-roGFP2 for chloroplast and mitochrondria targeting. After transforming GRX1-roGFP2, GFP fluorescence intensity was significantly changed by DTT (reductant) or H2O2 (oxidant) in rice protoplasts, which reflected the fluctuation of GSH redox status. The results showed that GRX1-roGFP2 could detect GSH redox changes in rice protoplast system. Application of protoplast system on the redox status of GSH in glutathione reductase 2 (GR2)-RNAi transgenic plants showed that GSH/GSSG ratio significantly decreased in cytosol by confocal microscopy and fluorescence microplate reader. Thus, transient expression of GRX1-roGFP2 in rice protoplasts for detecting GSH redox changes can be a convenient and efficient system for detecting the redox changes in plant organelles.