阿拉伯芥FAR-RED INSENSITIVE219與Cryptochrome2 的交互作用對藍光和茉莉酸訊息傳遞之整合的功能性研究

Functional Studies of ArabidopsisFAR-RED INSENSITIVE219 and Cryptochrome2 interaction in the integration of blue light and JA signaling

張紫煥(Tzu-Huang Chang)

指導教授 ： 謝旭亮

國立臺灣大學/生命科學院/植物科學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.02355

摘要

模式生物阿拉伯芥的幼苗會在有充足光照的情形呈現被稱為光型態發生(photomorphogenesis)的發育型態，而相對地在黑暗中呈現被稱為暗型態發生(skotomorphogenesis)的發育型態。根據目前的研究結果，我們了解光型態發生主要受到植物中的光接受體，尤其是吸收紅光及遠紅光波長的光敏素 (phytochrome)以及主要吸收藍光波長的隱花色素 (cryptochrome)所調控。Far-red insensitive 219 (FIN219)是與茉莉酸 (Jasmonate)訊息傳遞相關的酵素蛋白質，卻被報導能參與在受PHYA誘發的遠紅光訊息傳遞路徑當中。事實上後續的實驗證明大量表現FIN219功能性蛋白質的植物幼苗，在白光與藍光環境下都呈對光照過度敏感的外表型，這說明FIN219基因在這些光訊息傳遞路徑中可能也存在著某種角色。在前人的研究中指出，FIN219與CRY2不只在酵母菌雙雜交法中顯示有直接的交互作用，更在洋蔥表皮短暫表現法中顯示了他們在細胞內表現位置的重疊。為了進一步探索FIN219是否透過與CRY2的交互作用而影響藍光訊息傳遞，或是否CRY2也能透過FIN219而參與茉莉酸訊息傳遞，我們建立了大量表現CRY2蛋白質或是大量表現CRY2-C端片段的轉殖株。在後續的實驗中我們也將它們與FIN219基因相關的突變株與轉殖株雜交，並在所得到的子代中觀察FIN219與CRY2在這兩個訊息傳遞路徑上的交互影響。結果顯示，在藍光環境中FIN219參與在一個依賴於功能性CRY2蛋白質的下胚軸抑制路徑當中，並且FIN219具有正調控植物開花週期的功能。而我們同時也發現CRY2參與在由甲基茉莉酸 (MeJA)所觸發的抑制植物根及下胚軸生長的訊息傳遞路徑當中，並且由蛋白質表現量分析推測CRY2能藉由負調控FIN219的蛋白表現而造成這些現象。另外也觀察到CRY2能正調控花青素(Anthocyanin)的累積，而花青素的累積已知道是會受JA訊息傳遞所調控的。最後，藉由共免疫沉澱 (Co-immunoprecipitation)分析證實了在光照中植物體內FIN219與CRY2的直接交互作用。綜合目前的研究成果顯示，FIN219與CRY2能參與在彼此所屬的、並且截然不同的兩條訊息傳遞路徑當中，這樣的現象代表了植物具有整合各種環境刺激訊息的能力。

關鍵字

阿拉伯芥 ； 隱花色素2 ； 茉莉酸訊息傳遞 ； 藍光訊息傳遞 ； 遠紅光訊息傳遞

並列摘要

Arabidopsis thaliana seedlings undergo photomorphogenesis in the light condition and skotomorphogenesis 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/JAR1), a jasmonic acid (JA)-conjugating enzyme, was previously shown to be involved in phytochrome A (phyA)-mediated far-red light signaling pathway. However, the gain-of-function of FIN219 results in a hypersensitive phenotype in response to far-red, blue and white light, which suggests that FIN219 might play a role not only in far-red light but also in blue light signaling. Previous studies showed that Arabidopsis cryptochrome 2 (CRY2) physically interacted with FIN219 in vitro. To further elucidate the physiological functions of FIN219 and CRY2 interaction in blue light signaling and to know whether CRY2 can also participate in JA signaling through the interaction with FIN219, we generated transgenic lines harboring CRY2 and CRY2-Cterminal (CCT2) overexpression and built the crossed-plant materials between these CRY2-associated and FIN219-associated transgenic lines to observe their phenotypic responses under different light and different MeJA treatment conditions. The results revealed that FIN219 requires a functional CRY2 to regulate hypocotyl elongation under blue light and that FIN219 is a positive regulator of Arabidopsis flowering under long-day condition.Moreover, CRY2 is involved in MeJA-mediated inhibition of hypocotyl and root elongation likely throughinhibiting the expression level of FIN219 protein. Besides, CRY2 can positively regulate the anthocyanin level that is also regulated by MeJA.Finally, we demonstrated the physical interaction between FIN219 and CRY2 in vivo by using the Co-immunoprecipitationassay . Taken together, our data reveal that FIN219 and CRY2 are able to regulate the responses of the BL and JA signaling pathways, which may represent the integrated point where JA signaling and blue light signaling intersect. In the future, we will further elucidate the molecular mechanisms underlying their integration.

並列關鍵字

Arabidopsis ； Cryptochrome2 ； JA signaling pathway ； FR light signaling pathway ； Blue light signaling pathway

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