阿拉伯芥中FIN219和光敏素A交互作用之研究

Abstract

光敏素A是阿拉伯芥細胞中負責接收遠紅光的光接受者，能接收波長介於700-750 nm 區間的光源。在先前的報導中指出，FIN219是參與在光敏素A所引導的遠紅光訊息傳遞路徑中的一個正向傳遞因子，當FIN219此基因突變時，阿拉伯芥的幼苗會對遠紅光產生不敏感的外表型，呈現出一個長下胚軸的外型。然而目前對於光敏素A和FIN219在植物細胞中的調控機制並不清楚。在蛋白質表現的實驗分析中，發現將黑暗中生長的幼苗短暫地以遠紅光照射時，FIN219的蛋白質表現量會受到遠紅光的刺激而大量表現。除此之外，在不同的光敏素A突變株中，FIN219的蛋白質表現量會下降，暗示FIN219會受到光敏素A的正向調控。利用酵母菌雙雜交的方法，顯示光敏素A和FIN219 彼此之間具有交互作用的關係；此交互作用關係也利用體外結合能力測試的方法再次加以證明。另一方面，FIN219能和Pr及Pfr形式的光敏素A結合，而對Pr形式的光敏素A具有較高的結合能力。透過阿拉伯芥的原生質體來觀察FIN219在細胞內的位置，顯示FIN219蛋白質大量地累積在細胞質裡；同時亦可累積在細胞核中。在fin219及光敏素A的雙突變體的外表型分析中，發現在遠紅光生長的環境下，fin219及光敏素A的雙突變植株幼苗的下胚軸長度會比fin219或光敏素A的單一突變植株還要來的長，意味著當植物面對遠紅光的反應時，光敏素A會結合FIN219來調控植株幼苗下胚軸的延長。此外，藉由RT-PCR的分析也顯示出一些受光調控的基因在fin219及光敏素A的雙突變體中，會受到不同差異程度地影響。在藉由轉殖品系的分析中，發現同時具有大量表現FIN219 C端274個氨基酸和大量表現光敏素A的N端的轉殖株，會呈現出對遠紅光不敏感的外表型，暗示FIN219會和光敏素A結合來影響到遠紅光的訊息傳遞。觀察此轉殖雜交品系的開花時間，發現在長日照的環境條件下會有延遲開花的現象。同時，我們也發現在植物細胞中FIN219可能以一個磷酸化蛋白質的形式存在，而去磷酸化之後的FIN219會變得不穩定。然而FIN219是否為光敏素A的受質目前並不清楚。

Phytochrome A (phyA) is the photoreceptor of far-red light (FR) (700-750nm) in Arabidopsis. Far-red insensitive 219 (FIN219) has been shown to be a positive signal component in phyA-mediated FR signaling pathway, and fin219 mutant exhibits a long hypocotyl phenotype under FR. How phyA regulates FIN219 remains unknown. Protein gel blot analyses indicated that FIN219 protein was induced once exposure to FR from darkness. Besides, FIN219 was decreased in different phyA alleles, indicating that phyA positively regulates FIN219. Yeast-two-hybrid assays revealed that FIN219 can interact with PHYA, and this interaction was further confirmed by in vitro pull-down assay. Moreover, FIN219 can bind with both Pr and Pfr forms of phyA, but with higher affinity with Pr form. Subcellular localization studies in Arabidopsis protoplasts indicated that FIN219 accumulated in the nuclei, in addition to the cytoplasms. In addition, the hypocotyl length of fin219/phyA double mutant was longer than that of fin219 or phyA single mutant under FR, implying that FIN219 and phyA bind together to regulate hypocotyl elongation in response to FR. Furthermore, RT-PCR assays revealed that some light-regulated genes were differentially affected in fin219/phyA. Transgenic seedlings, FIN219-C274/PHYA-N, harboring the overexpression constructs of the C-terminal 274 amino-acid region of FIN219 and the N-terminal domain of PHYA exhibited a hyposensitive phenotype under FR, suggesting that the interaction between FIN219 and phyA affects FR light signaling. This transgenic line FIN219-C/PHYA-N also revealed a delayed- flowering phenotype under long-day condition. As well, we found that FIN219 may exist as a phosphorylated protein in vivo, and the unphosphorylated form of FIN219 is not stable. Whether FIN219 is a substrate of phyA remains to be elucidated.