Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90090
標題: Characterization of AtRboh I under Hypoxic Stress in Arabidopsis
阿拉伯芥中 AtRboh I 在缺氧逆境下 之特性分析
作者: 林依萱
I-Shiuan Lin
關鍵字: 阿拉伯芥
缺氧逆境
NADPH氧化酶
生長素
Aradopsis
hypoxia
rboh I
NADPH oxidase
auxin
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摘要: 當植物遭受環境逆境時,其體內之荷爾蒙及氧化還原狀態之平衡皆受到 影響,以使其能因應逆境得以生存,在此過程中,往往伴隨著活性氧族 (Reactive oxygen species, ROS)的產生。高等植物可透過 NADPH 氧化酶之作 用 生 成 超 氧 陰 離 子 (superoxide anion radical, O2.-) 進 而 產 生 過 氧 化 氫 (hydrogen peroxide, H2O2)等活性氧族,NADPH 氧化酶又稱為 RBOHs (respiratory burst oxidase homologues)。前人研究顯示,阿拉伯芥中十個 Rboh 基因家族成員中,有五個成員基因表現會受缺氧逆境所誘導,分別為 AtRboh A、B、D、G 及 I,其中 AtRboh I 轉錄子在缺氧逆境下 3 小時開始受誘導, 到 9 小時達到最高量。為深入探討缺氧訊號路徑下 AtRboh I 之功能,我們 利用阿拉伯芥 T-DNA 插入之 rboh I 突變株與野生型,進行生理及分子特性 分析。在缺氧逆境下之存活率結果顯示,rboh I 突變株之存活率低於野生型。 葉綠素含量試驗顯示,回復缺氧逆境處理後,rboh I 突變株幼苗葉綠素含量 較野生型低。利用 AtRboh I pro ::GUS 轉殖株偵測 AtRboh I 啟動子在不同組織 之表現情形,發現 AtRboh I 啟動子表現於幼苗之根部維管束組織、葉脈、毛 狀體、花柱頂部及果莢兩端,並受機械損傷所誘導表現。GUS 活性分析結 果 顯 示 , 受 缺 氧 逆 境 誘 導 之 GUS 活 性 在 添 加 生 長 素 運 移 抑 制 劑 (1-naphthylphthalamic acid , NPA)處理下,誘導情形會受抑制。進一步利用即 時定量聚合酶連鎖反應 (real-time quantitative polymerase chain reaction, q-PCR)分析缺氧訊號、乙烯生合成之相關基因及生長素反應基因,結果顯示 在缺氧逆境下,缺氧訊號相關基因 AtHRE1 、 AtADH1、AtLDH 及 AtSUS1 在 rboh I 突變株中之表現量皆下降,而在添加 NPA 處理下,僅 AtSUS1 之表現 量上升; 而在缺氧逆境下,生長素反應基因 At1g19840、At3g23030 及 At5g19140 表現量在 rboh I 突變株中之表現量皆上升;在缺氧逆境並添加 NPA 處理下,乙烯生合成之關鍵酵素基因 AtACS7 及 AtACS8 於 rboh I 突變 株中之表現量皆上升。綜合以上實驗結果顯示,於缺氧逆境下,AtRboh I 參與調控乙烯生合成及缺氧相關基因之表現,及缺氧訊號下與生長素訊號路 徑之交互作用,進而影響植株之缺氧耐受性。
Environmental stresses can cause accumulation of reactive oxygen species (ROS) in higher plants which have developed adaptive mechanisms by influence homeostasis of redox state and phytohormones. The NADPH oxidase family lead to the production of apoplastic superoxide (O2.–), then rapidly catalyzed to hydrogen peroxide (H2O2). The NADPH oxidase has ten known members in Arabidopsis which is named respiratory burst oxidase homologues (AtRboh A-J). The transcript levels of five AtRboh genes (AtRboh A, B, D, G and I) were increased under hypoxia. In particular, the transcript levels of AtRboh I were induced after 3 h hypoxia treatment and highest transcript levels after 9 h hypoxia treatment. To further investigate the function of AtRboh I under hypoxic signaling pathways, we used two independent AtRboh I-knockout lines to assess molecular function in Arabidopsis thaliana. Under submergence condition, the AtRboh I displayed reduced survival rate compared with wild-type. Furthermore, the chlorophyll content of AtRbohI-knockout lines displayed lower than that detected in wild-type. Histochemical analysis results presented that GUS expression was detected at vascular tissue of roots and leaves, trichomes, top of column and both ends of siliques in AtRboh I pro ::GUS transgenic plants under normoxic condition, also induced by wounding treatment. The GUS activity assay was showed that AtRboh I promoter was induced by hypoxia, but reduced in hypoxia combined with auxin transport inhibitor (1-naphthylphthalamic acid, NPA) treatment. Quantitative-PCR analyses were showed that hypoxia-inducible AtHRE1, AtADH1, AtLDH and AtSUS1 expression were reduced in AtRboh I-knockout lines under hypoxia, by contrast, AtSUS1 expression increased under hypoxia combined with NPA treatment. Expression of auxin responsive genes At1g19840, At3g23030 and At5g19140 were increased under hypoxia. Moreover, expression of the ethylene biosynthetic genes AtACS7 and AtACS8 were increased under hypoxia combined with NPA treatment. Taken together, our results demonstrate that AtRboh I plays an important role in modulating genes expression of ethylene biosynthesis and down-stream of hypoxia signaling, and interplays of hypoxia signaling and auxin-mediated signaling pathways under hypoxic stress, to give further effect on hypoxia tolerance in plants.
URI: http://hdl.handle.net/11455/90090
文章公開時間: 2018-07-29
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