Glutathione Reductase 3 對水稻鹽分逆境功能之研究

Abstract

鹽分逆境常誘導活化氧族（reactive oxygen species , ROS）產生，對植物造成氧化傷害，植物為了降低鹽分逆境下的氧化傷害發展出許多應對的策略，如ascorbate-glutathione cycle（ASC-GSH cycle），其中 glutathione reductase（GR）在 ASH-GSH cycle 中扮演重要角色，負責將氧化態的GSSG 還原為GSH。水稻GR 基因家族由三個基因組成，分別為細胞質的OsGR2，和葉綠體的 OsGR1及OsGR3。我們過去的研究發現 OsGR3 在水稻幼苗根部受 NaCl 和 abscisic acid 誘導表現，但是其胺基酸序列由於缺失了重要的 FAD 結合功能區塊，被認為是一個不具有功能的 GR 同功酵素。本試驗透過水稻序列資料庫比對 OsGR3 序列後，發現在預測序列 N 端應存在一個 FAD 結合功能區塊，為進一步了解此一全長的 OsGR3 是否具有GR 活性，及鹽分逆境對 OsGR3 的反應，本試驗透過大量表現 OsGR3 及osgr3 突變體研究其功能。結果顯示， osgr3 突變株會降低 42% 的 GR 活性，同時幼苗表現出對鹽分敏感的外表型，處理鹽分一週後 osgr3 存活率僅為 9%，在第二片葉片會累積較多的 H2O2，並降低 Fv/Fm。進一步分析發現，osgr3 幼苗在鹽分逆境下，ascorbate （ASC） 含量減少，且ASC/DHA（dehydroascorbate）降低，推測這可能是導致 osgr3 不耐鹽分逆境之原因。在大量表現 OsGR3 植株中，GR 活性提高 3∼6 倍，並比 WT 有較高鹽分耐受性，且大量表現 OsGR3 之轉殖株含有較高的 ASC 含量。而在大量表現 FAD 缺失的OsGR3tc 轉殖株中，GR 活性降低，且植株不耐鹽。另一方面，將 OsGR3 轉殖回 osgr3 的功能補償轉殖株則可增加 3∼5 倍 GR 活性降低，並回復對高鹽逆境的耐受性。此外，以 methyl viologen (MV) 處理水稻葉圓片，結果顯示抵抗葉綠素降解能力以 osgr3 功能補償植株最佳，其次為 WT，而以 osgr3 居於最後，顯示OsGR3和水稻抗氧化逆境有關。綜合以上結果，OsGR3不但具有 GR 活性，並與水稻的鹽分耐受性及抗氧化逆境有關。

Salt stress results in an excessive generation of reactive oxygen species (ROS) that causes oxidative damage to plants. Plants evolve certain strategies to remove salt-induced reactive oxygen species (ROS), such as glutathione-ascorbate cycle (ASH-GSH cycle). Glutathione reductase (GR) plays an important role in ASC-GSH cycle, which converted oxidized GSH (GSSG) to reduced GSH. Three GR genes exist in rice, including a cytosolic (OsGR2) and two chloroplastic isoforms (OsGR1 and OsGR3). Our previous study demonstrated that expression of OsGR3 was increased in response to NaCl and abscisic acid in roots of etiolated rice seedlings. The OsGR3 has been considered to be a non-functional GR isozyme due to the lack of a FAD binding domain and chloroplastic transit sequence (CTS). However, after blasting different rice database, we found a full length OsGR3 open reading frame with complete FAD binding domain and CTS. To investigate the physiological roles of OsGR3 in salt stress and to understand whether the OsGR3 is a functional GR, OsGR3 knockout mutant and overexpression transgenic rice were analyzed in this thesis. In osgr3, 42% GR activity was decreased and salt sensitivity was enhanced. Physiological responses of osgr3 rice mutant revealed that the maximal efficiency of photosystem II and survival rate were notably reduced as compared to WT, and H2O2 was increased after 200 mM NaCl treatment. One of the reasons caused osgr3 mutant intolerant to salt stress is that osgr3 had lower ascorbate content and lower ratio of ascobate/dehydroascorbate than WT rice. Overexpression of OsGR3 in rice plants increased three to six times of GR activity, and also increased transformant's salt tolerance. Higher ascorbate content in overexpression OsGR3 transgenic rice as compared to WT was also observed. However, overexpression of OsGR3tc, lack of FAD binding domain, diminished GR activity, and transgenic rice were sensitive to salt stress. On the other hand, complementation of osgr3 with OsGR3 (osgr3/OsGR3) increased three to five times of GR activity and restored salt stress tolerance. Meanwhile, leaf discs of osgr3, WT and complementation treated with methyl viologen showed that the degrees of chlorophyll bleaching from osgr3 to WT then osgr3/OsGR3, indicating that OsGR3 is important for oxidative defense system. Our results suggested that OsGR3 is a functional GR, and is involved in salt and oxidative tolerance.