水稻穀胱甘肽還原酶 1 調控側根發育之研究

Abstract

穀胱甘肽還原酶 (Glutathione reductase, GR) 為植物重要的抗氧化酵素，以 NADPH 為基質催化氧化態的 GSSG 還原為 GSH。水稻 GR 基因家族由三個基因組成，分別為位於細胞質的 OsGR2 和葉綠體/粒線體的 OsGR1 與 OsGR3。在 GR1::GUS 轉殖水稻中， GUS 主要表現於種子的胚與糊粉層、莖、葉鞘、葉片、花與根之維管束、側根原基與根尖。西方墨點法分析 OsGR1 對非生物性逆境之反應，發現水稻幼苗地上部OsGR1 為持續性表現，不受非生物逆境與植物荷爾蒙影響，反之，根部 OsGR1 則會受到 ABA、Auxin 與 Cytokinin 誘導表現。利用RNAi 技術弱化 OsGR1 表現，顯示 OsGR1 於地上部與根部分別貢獻40% 與 15% 的總GR 活性。弱化OsGR1 表現後會顯著抑制側根的發育，利用 GSH 專一性螢光探針，monobromobimane (mBBr) 偵測 GSH，發現 GR1-RNAi 轉殖株側根根尖的GSH 含量顯著降低，而外加 GSH 則可回復側根正常的生長。以受 Auxin 誘導之DR5::GUS 轉殖水稻進行組織化學染色分析，結果顯示 GSH 生合成抑制劑buthionine sulfoximine (BSO) 處理會降低側根中 GUS 的表現，並且隨著處理濃度增加抑制效果越顯著。此外，外加 GSH 也可減緩ABA 抑制側根發育與種子的發芽，並回復DR5::GUS 轉殖水稻側根中受ABA 抑制的 GUS 表現，分析ABRC::Luciferase 轉殖水稻，發現 GSH 可抑制 ABA 對ABRC 的誘導。另一方面，大量表現 OsGR1 可降低 ABA 對側根的抑制效果，維持側根根尖 GSH 含量的恆定，並可促進滲透壓逆境下側根的發育。以上結果說明 OsGR1 透過調控側根根尖 GSH 之穩定來調節水稻根部auxin 的轉運，與側根根尖對 ABA 的敏感度，進而調控側根的發育。

Glutathione reductases (GRs) are important antioxidant enzymes that catalyze the reduction of glutathione disulfide (GSSG) into reduced glutathione (GSH) with the accompanying oxidation of NADPH. In rice, one cytosolic (OsGR2) and two chloroplast / mitochondria dual-localized GR (OsGR1 and OsGR3) isoforms have been identified. In this study, role of the chloroplast / mitochondria localized OsGR1 was investigated. OsGR1 is primarily expressed in shoots at seedling stage and ubiquitously expressed in all tissues at heading stage. Transgenic rice containing GR1::GUS showed that GUS was expressed in vascular bundle and apical meristem of roots, embryo and aleurone layer of seeds, coleoptile, sheath, stem and leaves. Reduced expression of GR1 by RNA interference (GR1-RNAi) revealed that GR1 had contributed at least 40% and 15% of total GR activity in shoots and roots, respectively. Phenotypic analysis showed that lateral root (LR) growth was strongly inhibited in GR1-RNAi plants. Decreased glutathione levels was observed by GSH specific fluorescent-dye monobromobimane in LR-tip of GR1-RNAi plants, whereas exogenously applied GSH recovered the growth of LR. Treating DR5::GUS transgenic plant, an auxin indicator, with GSH biosynthetic inhibitor buthionine sulfoximine (BSO) showed that a decline in GUS expression was associated with increased BSO concentrations in LR; moreover, exogenously applied GSH relieved the inhibitory effect of ABA on LR development, seed germination, and GUS expression and of DR5::GUS transgenic plants. Through in vivo analysis of luciferase activity in ABRC::Luciferase transgenic rice showed that GSH retards ABA response and signal transduction in rice. In GR1-overexpressing plants, ABA inhibited LR growth was decreased and LR growth was improved under osmotic stress. Taken together, GR1-regulated GSH homeostasis in roots plays a key role in LR development by modulating auxin transport and the sensitivity to ABA.