維他命C及穀胱甘肽的氧化還原態對高溫誘導南西文心蘭開花的影響

文心蘭之抽梗不受季節調控，因此具潛力作為研究終年開花機制之模式植物。實驗室前人研究指出，低含量維他命C (抗壞血酸; ascorbate; AsA)為文心蘭開花之重要因子。然而，何種環境因子可有效控制文心蘭花期至今仍不明瞭。本研究發現，長時間高溫(30°C，14天以上)可有效誘導文心蘭開花。其花芽分化與高溫逆境相關，過程中可發現明顯被生合成的過氧化氫含量(Hydrogen peroxide; H2O2)，伴隨下降的維他命C氧化還原比例(AsA redox ratio)，以及大量被誘導細胞質抗壞血酸過氧化酶(cytosolic ascorbate peroxidase; cytAPX1)之基因表現。於高溫下比較阿拉伯芥大量表現文心蘭細胞質抗壞血酸過氧化酶之轉殖株，並測定其維他命C氧化還原比例之實驗結果中加以說明，長時間高溫誘導文心蘭開花，與細胞質抗壞血酸過氧化物酶所調控的維他命C氧化還原比例有關 (第二章)。然而，受維他命C氧化還原狀態所影響的蛋白至今仍不被明瞭。相較於穀胱甘肽(glutathione; GSH)，許多轉錄因子已被證明受穀胱甘肽氧化還原比而調控。實驗發現文心蘭細胞質抗壞血酸過氧化酶可與維他命C以及穀胱甘肽反應，並清除過氧化氫，而該特性不同於模式植物-阿拉伯芥。利用蛋白結構預測進一步了解文心蘭與阿拉伯芥細胞質抗壞血酸過氧化酶之差異，並找出三個可能為影響此特性的關鍵胺基酸。藉由點突變技術，證明此胺基酸為造成細胞質抗壞血酸過氧化酶可與穀胱甘肽結合的關鍵因子。分別將文心蘭以及阿拉伯芥細胞質抗壞血酸過氧化酶超表現於阿拉伯芥野生型，以及維他命C、脫氫抗壞血酸還原酶(dehydroascorbate reductase; DHAR)缺乏之突變株dhar1，並測定其穀胱甘肽氧化還原狀態及觀察開花時間。結果顯示，文心蘭可藉由細胞質抗壞血酸過氧化酶直接氧化穀胱甘肽，或是藉由維他命C-穀胱甘肽循環兩種方式，影響穀胱甘肽之氧化還原狀態，並可利用穀胱甘肽作為還原劑於維他命C缺乏的環境中 (第三章)。總結本實驗結果，細胞質抗壞血酸過氧化酶所直接調控的維他命C及穀胱甘肽氧化還原狀態為高溫誘導文心蘭開花的重要因子。

關鍵字

文心蘭；維他命C氧化還原比；高溫；細胞質抗壞血酸過氧化酶；開花；穀胱甘肽氧化還原比

並列摘要

The bolting time of the Oncidium is not season-dependent and so it is a useful year-round model system to study thermal-induced flowering mechanisms in planta. Previously, we reported that low ascorbate (AsA) content is essential for floral transition in Oncidium; however, the environmental factors governing floral initiation is not elucidated. The current study revealed that a prolonged treatment of elevated temperature (30°C over a 14-day period) induces floral transition. This floral induction in response to high-temperature was associated with a significantly increased ROS level and lowered AsA redox ratio, as well as up-regulated expression of cytosolic ascorbate peroxidase (cytAPX1). Transgenic Arabidopsis ectopically overexpressing Oncidium cytAPX1 (OgcytAPX1) displayed an early-flowering phenotype and low AsA redox ratio under high-temperature (Chapter 2). However, little is known in the AsA redox target proteins. Differently, several transcription factors are regulated by GSH redox ratio and trigger downstream signaling transduction. Oncidium cytAPX1 displays two substrate binding specificity by utilizing both AsA and GSH for scavenging H2O2, while Arabidopsis cytAPX1 (AtcytAPX1) is not. The site-directed mutagenesis in recombinant protein containing substitution(s) at amino acid residues of Pro63, Asp75, and Tyr97 were generated. In-gel activity assay showed that only the mutant containing three combined substitutions, Pro63Asp-Asp75His-Tyr97His, lost the GSH binding activity, indicating Pro63, Asp75 and Tyr97 together determine the conformational structure for GSH binding site. The comparative functional study was carried out by overexpressing OgcytAPX1 and AtcytAPX1 respectively in Arabidopsis. OgcytAPX1-OE Arabidopsis conferred earlier flowering in 30°C with lower level and redox ratio of GSH, comparing to AtcytAPX¬-OE Arabidopsis. OgcytAPX-OE in Arabidopsis dehydroascorbate reductase deficient mutant (dhar1) presented lower level and redox ratio in GSH, comparing to AtcytAPX¬-OE in dhar1, suggesting that the catalysis of GSH substrate by OgcytAPX is independent from DHAR activity. OgcytAPX-OE and AtcytAPX-OE in Arabidopsis ascorbate deficient mutant (vtc1), validated that OgcytAPX can potentially use GSH as substrate in AsA starvation condition to scavenge H2O2. These results provide the first evidence of the dual substrate binding specificities on AsA and GSH for OgcytAPX1 in Oncidium. The distinct physiological functions of OgcytAPX1 in promoting thermal-induced flowering process and enhancing stress tolerances in plants are elucidated (Chapter 3). Taken together, results from this investigation of the thermal-induced flowering mechanism indicated that the AsA and GSH redox ratio is a master-switch to mediate phase transition from the vegetative to reproductive stage.

並列關鍵字

ascorbate (AsA) redox ratio ； cytosolic ascorbate peroxidase 1 (cytAPX1) ； flowering ； glutathione (GSH) redox ratio ； high temperature ； Oncidium.

參考文獻

Alonso-Blanco, C., Aarts, M.G.M, Bentsink, L., Keurentjes, J.J.B., Reymond, M., Vreugdenhil, D., and Koornneef, M. (2009) What has natural variation taught us about plant development, physiology, and adaptation? Plant Cell 21: 1877-1896.

An, F.M., Hsiao, S.R. and Chan, M.T. (2011) Sequencing-based approaches reveal low ambient temperature-responsive and tissue-specific microRNAs in Phalaenopsis orchid. PLoS One 6: e18937.

Apel, K. and Hirt, H. (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annl Rev Plant Biol 55: 373-399.

Attolico, A.D. and De Tullio, M.C. (2006) Increased ascorbate content delays flowering in long-day grown Arabidopsis thaliana (L.) Heynh. Plant Physiol Biochem 44: 462-466.

Ausin, I., Alonso-Blanco, C., Jarillo, J.A., Ruiz-Garcia, L. and Martinez-Zapater, J.M. (2004) Regulation of flowering time by FVE, a retinoblastoma-associated protein. Nat Genet 36: 162-166.

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維他命C及穀胱甘肽的氧化還原態對高溫誘導南西文心蘭開花的影響

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