- 學位論文
阿拉伯芥FIN219在藍光及遠紅光訊息傳遞交互關係中的功能性研究
Functional studies of Arabidopsis FIN219 in cross-talks between blue light and far-red light signaling
摘要
阿拉伯芥的幼苗在光照中行使光形態發生而在黑暗中呈現暗形態發生（白化現象），光形態發生常是由吸收紅光及遠紅光的光敏素和吸收UV-A及藍光的隱花色素所共同調控。先前研究顯示FIN219為GH3基因家族成員之一，參與在光敏素A所負責的的遠紅光訊息傳遞中，並做為cop1的基因外抑制體，然而FIN219大量表現的轉殖株會顯現出對遠紅光、藍光以及白光皆有過度敏感的外表型，暗示FIN219除了參與遠紅光訊息傳遞外，可能也在藍光訊息傳遞中扮演某種角色。這裡我們證實了在試管內阿拉伯芥的隱花色素CRY1與其C端(CCT1)可與FIN219蛋白質具有直接的交互作用關係；在洋蔥表皮細胞的細胞內表現位置分析顯示FIN219可能同時存在於細胞核與細胞質，並且與CRY1, CRY2及COP1具有位置重疊的關係。此外FIN219的蛋白質在照射藍光後會被迅速地誘導表現，並且這樣的調控關係需要具有功能的CRY1存在。為了更深入地了解FIN219在藍光訊息傳遞中所負責的生理功能，我們將35S啟動子接上全長的FIN219後，轉殖進入各種藍光接受體突變株（包括cry1、cry2和cry1cry2）及GUS-CCT1轉殖株中。GUS-CCT1是過量表現β-葡萄糖醛酸苷酵素(GUS)與CCT1的融合蛋白質並具有在各種光環境中包括在黑暗中呈現出高度敏感的短下胚軸外表型的轉殖株。實驗結果顯示異位表現FIN219在cry1、cry2和cry1cry2中，並無任何顯著的外表型差異，意味著FIN219必須要CRY1及CRY2同時存在的情況下才能在藍光中調控下胚軸的延長。此外，過量表現c-myc-FIN219能在各種光環境下將GUS-CCT1轉殖株回復成為野生型的外表型；在針對FIN219-OE/GUS-CCT1轉殖株幼苗的免疫轉漬分析法中，在各種光環境下皆無法偵測到GUS-CCT1及c-myc-FIN219的蛋白質表現，相反地，在這些轉殖株幼苗中卻能偵測到GUS-CCT1及c-myc-FIN219的mRNA層級的表現，顯示GUS-CCT1與c-myc-FIN219兩者的融合蛋白具有相互拮抗地轉錄後修飾作用的調控關係，而這樣的調控關係導致了下胚軸回復成野生型的外表型。另外，我們也證實了在長日照的環境中FIN219在阿拉伯芥的調控開花機制中是一個正向的調控者。綜合上述結果顯示FIN219在調控去白化現象及開花時間中所扮演的角色，很可能是在遠紅光訊息傳遞與藍光訊息傳遞網絡的交會點上。
並列摘要
Arabidopsis thaliana seedlings undergo photomorphogenesis in the light and skotomorphogenesis (etiolation) in the dark. Photomorphogenesis is often regulated by red/far-red light-absorbing phytochromes and UV-A/blue light-absorbing cryptochromes. Far-red insensitive 219 (FIN219), a member of GH3 gene family, was previously shown to be involved in phytochrome A (phyA)-mediated far-red light signaling acting as an extragenic suppressor of constitutive photomorphogenic 1 (cop1). However, the gain of function of FIN219 exhibits a hypersensitive phenotype in response to far-red, blue and white light, which suggests that FIN219 might play a role in blue light signaling in addition to far-red light. Here we show that Arabidopsis cryptochrome 1 (CRY1) and its C-terminal domain (CCT1) physically interact with FIN219 protein in vitro. Subcellular localization studies in onion (Allium cepa) epidermal cells reveal that FIN219 localizes in both the nucleus and the cytosol and co-localizes with CRY1, cryptochrome 2 (CRY2) and COP1. In addition, FIN219 was rapidly induced by blue light treatment and this induction required functional CRY1 in vivo. To further elucidate the physiological functions of FIN219 in blue light signaling, a full-length FIN219 construct driven by 35S promoter was introduced into the blue light photoreceptor mutants, cry1, cry2 and cry1cry2, as well as GUS-CCT1 transgenic plants that overexpressed β-glucuronidase (GUS) and CCT1 fusion proteins showing a hyperphotomorphogenic development under all light conditions, including the darkness. The results revealed that ectopic expression of FIN219 in Arabidopsis blue light receptor mutant backgrounds, cry1, cry2 or cry1cry2, did not result in obvious phenotype, which implies that FIN219 requires the presence of CRY1 and CRY2 to regulate hypocotyl elongation under blue light. Moreover, GUS-CCT1 transgenic plants can be rescued by overexpression of a c-myc-FIN219 construct compared to wild-type phenotype under various light conditions. Nevertheless, in immunoblot analysis of FIN219-OE/GUS-CCT1 transgenic seedlings, we found that GUS-CCT1 and c-myc-FIN219 were hardly detectable. In contrast, GUS-CCT1 and c-myc-FIN219 mRNA transcripts in rescued seedlings can be detected, suggesting that c-myc-FIN219 and GUS-CCT1 fusion proteins might be antagonistically regulated in posttranscriptional modifications, leading to a rescued hypocotyl phenotype. Finally, we demonstrate that FIN219 is a positive regulator of Arabidopsis flowering under long-day condition. Taken together, the roles of FIN219 in regulating both of the de-etiolation and flowering responses may represent the integrated point where far-red light signaling and blue light signaling intersect.