GST成員參與阿拉伯芥光訊息傳遞的研究

植物多為固著生長，行光合作用，因此因應外界光線環境的改變，而調整自身的生長與發育能力，對植物尤其重要。在阿拉伯芥中，光敏素 A是負責感受光環境中的遠紅光，並透過一連串的訊息傳遞而影響下游的基因表現，而使植物的光型態（photomorphogenesis）發生。FIN219是一個已被證實參與在遠紅光訊息傳遞上游因子（Hsieh et al., 2000）。一個產生GST(glutathione S-transferases)蛋白質基因FIP1(FIN219-interacting protein 1)，它所產生的蛋白質被證實可與FIN219的C端作用 (黃怡靜論文，2003)。然而，Tepperman et al.(2001)利用microarry方式也檢測到一個GST(AtGSTU17)可受到遠紅光的誘導，但此mRNA表現量的增加受到光敏素A突變體的抑制。因此，是否部分GST基因家族成員參與光訊息傳遞與植物生長與發育，乃是個有趣的研究主題。利用phyA,fin219突變體進行RT-PCR與Northern blotting分析發現，AtGSTU17在遠紅光下的表現確實受到phyA與fin219突變的抑制。其mRNA的表現亦隨著光照時間越長，基因表現越強，在遠紅光照射六個小時，基因表現達到最高峰；且隨著遠紅光強度越強，AtGSTU17也表現越強。此結果顯示AtGSTU17的表現與遠紅光光照有密切的關係。將AtGSTU17由阿拉伯芥中釣取出，將其轉入大腸桿菌中，純化出的AtGSTU17蛋白質，是具有GST酵素活性，證實AtGSTU17的確是一個GST，且具有受光調控的功能。大量表現AtGSTU17在野生型阿拉伯芥中，遠紅光中生長的轉殖株幼苗，會有比野生型更短的下胚軸長度，其葉綠素、花青素的累積會受到影響；從ABRC獲得的T-DNA 插入品系，在遠紅光下的突變株幼苗，會有比野生型更長的下胚軸外表型，開花時間有延遲的現象；其葉綠素、花青素的累積也會受到影響，與大量表現AtGSTU17的轉殖株幼苗具有相反的外表型，由此可知AtGSTU17是參與在遠紅光下的光型態發生。除此之外，利用AtGSTU17的啟動子與GUS的轉錄融合體來探討AtGSTU17基因的表現機制。結果顯示在黑暗、遠紅光、藍光、紅光下生長四天大的植株，主要皆表現在維管束組織，三十天大的植株GUS表現在花柱、雄蕊、花萼、花瓣的維管束及種子柄，顯示ATGSTU17可能作用在此部位進而影響植物生長與發育。利用突變株雜交測試，初步由F1世代在遠紅光下有長下胚軸外表型，且比個別突變株在heterozygote時的下胚軸長，顯示PHYA與ATGSTU17兩者之間具有非等位非互補（nonallelic-noncomplementation）的遺傳調控關係，可能共同參與在訊息傳遞路徑。此外，Northern blotting分析發現AtGSTU17在白光下的表現會受到離層酸、生長素2,4-D與茉莉侗酸(jasmonic acid)的誘導；且在ABA抑制根生長實驗中，T-DNA突變株展現一個比野生型還不敏感的外表型。綜合上述結果顯示AtGSTU17是個GST基因家族成員，它的表現會受到PHYA與不同賀爾蒙的調控，以及光強度的影響，並且會影響植物在遠紅光下的光型態發生及賀爾蒙控制的根的延長。

關鍵字

阿拉伯芥；光型態發生

並列摘要

Previous studies have shown that a glutathione S-transferase (GST), a tau class member of the GST gene family in Arabidopsis, can interact with the C-terminus of FIN219 that functions as a positive regulator of phytochrome A (phyA)-mediated far-red (FR) light signaling. Tepperman et al. (2001) reported that a GST (AtGSTU17) mRNA expression was induced rapidly in wild type under FR light, but abolished in the phyA mutant. To further investigate whether some GST members will be involved in photomorphogenesis, we analyzed the AtGSTU17 transcript under FR condition. Northern blotting analyses showed that AtGSTU17 expression was indeed induced by FR and regulated by PHYA. Moreover, its expression was light intensity-dependent. Purified recombinant protein AtGSTU17 also showed GST activities with both substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Furthermore, transgenic seedlings overexpressing AtGSTU17 exhibited a hypersensitive phenotype under FR, whereas the knock-out mutant of AtGSTU17 (atgstu17) showed a longer hypocotyl phenotype compared to wild type. In addition, transgenic seedlings overexpressing or inhibiting AtGSTU17 expression also affected FR-mediated anthocyanin accumulation and chlorophyll killing. Moreover, atgstu17 mutants displayed a delayed flowering phenotype under long-day condition. In contrast, 35S:AtGSTU17 transgenic plants exhibited slightly earlier flowering under the same condition. Genetic analysis indicated that PHYA and ATGSTU17 appeared to show a relationship of nonallelic noncomplementation. Promoter activity assays revealed that AtGSTU17 was mainly expressed in vascular tissues of the seedlings and floral organs. As well, AtGSTU17 was induced by several hormones, such as ABA, 2,4-D, and JA. Intriguingly, atgstu17 seedlings displayed insensitivity to ABA-mediated inhibition of root elongation, whereas, 35S:AtGSTU17 transgenic plants showed almost equal sensitivity to exogenous ABA effect. Taken together, our data indicate AtGSTU17 may function as a crosstalk between FR light and multiple hormones to regulate hypocotyl elongation and root growth.

並列關鍵字

GST ； PHOTOMORPHOGENESIS

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GST成員參與阿拉伯芥光訊息傳遞的研究

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