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標題: Characterization of RbohD/ein2-5 double mutant under Hypoxia Stress
作者: 洪晨溥
Chen-Pu Hong
關鍵字: ethylene;hydrogen peroxide;hypoxia;乙烯;過氧化氫;缺氧
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植物荷爾蒙乙烯 (ethylene, ET),已被證實參與在高等植物遭遇淹水逆境時所導致的缺氧訊號傳遞路徑中。研究發現除了乙烯,過氧化氫 (hydrogen peroxide, H2O2)亦會在缺氧訊號下被累積,而過氧化氫之生成,可由位於細胞膜上之NADPH氧化?所產生。NADPH氧化?作用會生成超氧陰離子 (superoxide anion radical, O2-),且迅速被超氧化物歧化? (superoxide dismutase, SOD)氧化為過氧化氫。高等植物中NADPH氧化?又稱為respiratory burst oxidase homolog (Rboh)。為深入探討缺氧訊號路徑下,乙烯與過氧化氫交互作用之影響,本實驗利用阿拉伯芥野生型、ein2-5乙烯不敏感突變株、rbohD突變株及rbohD/ein2-5雙突變株進行缺氧環境下之生理及分子特性分析。種子萌發試驗結果顯示rbohD/ein2-5雙突變株具延遲種子萌發之現象,且rbohD/ein2-5雙突變種子萌發後幼苗生長時期根之生長速率較野生型快。缺氧處理下3,3'-diaminobenzidine (DAB)染色結果顯示,rbohD/ein2-5雙突變株內過氧化氫累積較野生型少,且rbohD/ein2-5雙突變株幼苗之根長未如野生型呈現根長受抑制之現象。葉綠素試驗顯示,缺氧訊號下rbohD/ein2-5雙突變株幼苗葉綠素含量較野生型低。進一步利用即時定量聚合?鏈鎖反應 (real-time quantitative PCR)分析結果發現,受缺氧所誘導之hypoxia responsive element 1 (AtERF73/HRE1)與alcohol dehydrogenase 1 (ADH1)表現量,在rbohD/ein2-5雙突變株中誘導表現量較野生型、rbohD單突變株及ein2-5單突變株低。進一步利用RbohDpro::GUS轉殖株偵測RbohD啟動子在不同組織中之表現情形,發現RbohD啟動子表現於幼苗根部維管束與葉片之部位。綜合以上實驗結果顯示,於正常生長情況下乙烯與過氧化氫之交互作用影響種子萌發與萌發後根之生長速率,於缺氧逆境下參與調控幼苗根之生長、葉綠素含量及缺氧相關基因之表現。

Under conditions of limited oxygen availability (hypoxia), the phytohormone ethylene is involved in hypoxia signalling pathway in higher plants. Previous research in our laboratory showed that in addition to ethylene, the hydrogen peroxide (H2O2) was also accumulated under hypoxic stress which was produced by the potential source of NADPH oxidase. The NADPH oxidase family is localized in the plasma membrane and transfers electrons from cytosolic NADPH or NADH to apoplastic oxygen, leading to the production of apoplastic superoxide. The SODs rapidly catalyze the production of H2O2 from superoxide. The functional relationship between ethylene and H2O2 in the hypoxia signaling mechanism is still poorly understood. To further investigate the interplay of ethylene and H2O2 under hypoxia signalling pathway, we use Arabidopsis thaliana ecotype Columbia (Col-0), the ethylene-insensitive mutant ein2-5 (Col-0), the RbohD (At5g47910) T-DNA insertion mutant and rbohD/ein2-5 double mutant was used to further physiological characterization and molecular analysis under hypoxic stress. Results from seeds germination and post-germination of seedling growth showed that the rbohD/ein2-5 double mutant was presented delay seeds germination but the root growth rate was faster compare with wild-type under normoxia condition. The 3,3'-diaminobenzidine (DAB) staining showed that the rbohD/ein2-5 double mutant was presented lower H2O2 accumulation compare with wild-type under hypoxia condition. The roots growth of rbohD/ein2-5 double mutant was not inhibited after submergence treatment. Furthermore, the level of chlorophyll content of rbohD/ein2-5 double mutant was display lower than wild-type after submergence treatment. The quantitative RT-PCR analysis was shown that induction level of AtERF73/HRE1 and AtADH1 transcripts was lower in rbohD/ein2-5 double mutant during hypoxic stress compare with wild-type, RbohD knockout mutant and ein2-5. The GUS expression was detected in the seeding vasculature and leaves in RbohDpro::GUS transgenic plants. Taken together, our results show that the interplay of ethylene and H2O2 influence seeds germination and post-germination stage under normoxia condition. Moreover, ethylene and H2O2 are involved in modulating root growth of seedlings, chlorophyll content of leaves and hypoxia-inducible genes expression under hypoxia condition.
其他識別: U0005-2811201416181338
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