穀胱甘肽的氧化與還原狀態在維他命C-穀胱甘肽循環鏈中與文心蘭開花機制之關聯性

Abstract

文心蘭生長過程中是如何決定走向營養生長或生殖生長之機制至今仍不明瞭。前人研究指出，在高溫 (30℃) 狀態下能促進文心蘭細胞質中之維他命C過氧化酶 (Oncidium cytosolic ascorbate peroxidase 1，OgcytAPX1) 將維他命C (ascorbic acid, AsA) 氧化，而降低維他命C之氧化還原比例 (還原態/氧化態)，促使文心蘭南西品系 (Oncidium Gower Ramsey) 提早開花。然而維他命C除了作為植物體最主要之抗氧化物及參與荷爾蒙生合成之輔酶等功能之外，目前並無研究報導指出其是否有確切之目標蛋白以及其是否能調控轉錄因子。為了瞭解維他命C氧化還原變化之訊息是如何傳遞至開花路徑，本研究發現，另一抗氧化物穀胱甘肽 (glutathione, GSH) 能將維他命C氧化還原比例變化之訊息傳遞至開花路徑，並且是藉由維他命C -穀胱甘肽循環 (AsA-GSH cycle) 路徑所致。藉由分析阿拉伯芥大量表現OgcytAPX1之轉殖株及其突變株，於高溫誘導下，間接探討維他命C與穀胱甘肽之上下游關係；結果顯示大量表現文心蘭細胞質維他命C過氧化酶之轉殖株，其穀胱甘肽含量及穀胱甘肽氧化還原比例會隨高溫處裡時間增加而顯著的降低，此表示OgcytAPX1能影響維他命C氧化還原比例進而影響穀胱甘肽氧化還原比例。利用生理生化分析測定穀胱甘肽氧化態與還原態，結果顯示，穀胱甘肽氧化還原比例於文心蘭開花時期會顯著的降低至一門檻值。進一步測定影響穀胱甘肽氧化態及還原態之相關基因之表現量以及其酵素活性，顯示榖胱甘肽生合成基因以及穀胱甘肽還原酶基因皆可在長時間高溫誘導下顯著的降低表現，使得氧化還原態比例降低。此結果說明穀胱甘肽氧化還原態比例與文心蘭開花誘導之機制有關。藉由外加處裡穀胱甘肽生合成抑制劑 (BSO) 於文心蘭，可發現文心蘭具有提早開花之表現型，並且分析文心蘭開花相關基因表現，GIGANTEA (GI), FLOWERING LOCUS T (FT), LEAFY (LFY), 及 APETALA 1 (AP1)，均可於處裡BSO及GSSG (氧化態之GSH) 之後有顯著提高表現，更加證明穀胱甘肽氧化還原態確實能夠影響文心蘭開花。總結上述結果，穀胱甘肽氧化還原態能將上游維他命C氧化還原之訊息傳遞至下游開花路徑，並且可能透過GI影響文心蘭開花路徑。

The mechanism of how the phase transition from vegetative to bolting stage in Oncidium is still not distinctive. Previous studies have shown that high temperature-induced flowering is regulated by Oncidium cytosolic ascorbate peroxidase 1 (OgcytAPX1) through mediating the redox state of ascorbate (AsA). However, there were no reports on the regulating proteins interacting with AsA in the flowering process. Here, we demonstrate that the effect of AsA redox state underlying flowering induction of Oncidium was correlated with glutathione (GSH) redox state, and this signal transduction is transduced via AsA-GSH cycle. By analyzing over-expression of OgcytAPX1 in Arabidopsis, and Arabidopsis mutant lines, apx1-1, to realize the relationship in redox homeostasis between AsA and GSH under high-temperature. This result indicate that the redox changes in GSH is closely related with AsA redox state, and further to induce flowering. Biochemical assays of the GSH levels, and GSH redox ratio, revealing that the decrease of GSH redox ratio was associated with bolting stage and high-temperature induced flowering. Analysis of transcripts level and enzymatic activity revealed that the GSH biosynthesis enzyme (γ-glutamylcysteine synthetase, GSH1 ; glutathione synthetase, GSH2) and glutathione reductase (GR1), the GSH-redox related enzyme, were decrease during bolting stage and after the high-temperature stress condition. To reconfirm the influence of GSH redox ratio in bolting, we applied glutathione biosynthesis inhibitor, buthionine sulphoximine (BSO), and GSSG (the oxidized form of GSH) in Oncidium. The results demonstrate that GSH redox ratio could strongly influence bolting. Moreover, analysis on the expression profiling of floral genes also demonstrating that GIGANTEA (GI), FLOWERING LOCUS T (FT), LEAFY (LFY), and APETALA 1 (AP1) are highly induced in these treatment, suggesting that GSH redox ratio plays the crucial role on flowering induction in Oncidium.