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標題: | 阿拉伯芥中的去磷酸酶調控FIN219/JAR1在光和茉莉酸訊息傳遞中的鑑定與功能性研究 Identification and functional studies of phosphatases regulating FIN219 in the integration of light and jasmonate signaling in Arabidopsis |
作者: | Yun-Ju Shyu 徐韻茹 |
指導教授: | 謝旭亮(Hsu-Liang Hsieh) |
關鍵字: | 藍光光型態發生,FIN219,茉莉酸,磷酸化修飾,去磷酸?, blue light photomorphogenesis,FIN219,jasmonate,phosphorylation,phosphatase, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 阿拉伯芥植物幼苗的光型態發生受到荷爾蒙與光受體調控。透過後轉譯修飾改變蛋白質的磷酸化狀態,以反應不同的周圍環境訊號。先前研究中, FIN219/JAR1催化茉莉酸(jasmonate)和異白胺酸(isoleucine)結合,使茉莉酸轉變為更具有生物活性的狀態。藍光下,酪蛋白激酶(CK2)會磷酸化FIN219/JAR1。而黑暗轉移到藍光的過程中,FIN219/JAR1的磷酸化狀態會有所改變,依此推測有去磷酸酶參與在其中,藉由調控FIN219/JAR1磷酸化狀態而影響光和茉莉酸反應。本研究透過兩途徑,Co-IP/ LC-MS/MS分析和受藍光調控表現的基因,挑選出兩個可能的候選去磷酸酶,EGR2和PP2CR。利用BiFC (Bimolecular Fluorescence Complementation)方法驗證,在黑暗和藍光情況下,此EGR2和PP2CR皆會與FIN219/JAR1有交互作用。藉由磷酸標籤蛋白質電泳,在EGR2和PP2CR突變的植株中,FIN219/JAR1呈現較磷酸化並且不穩定。EGR2突變植株對於藍光抑制下胚軸延長的外表型上較不敏感。處理茉莉酸後,egr2突變植株中花青素累積量較野生型多,pp2cr突變植株中花青素與葉綠素累積量皆相較於野生型少。並且基因表現層次上,短暫照射藍光30分鐘後,egr2突變植株中茉莉酸相關基因表現量較野生型高,光相關基因表現量較野生型低。在黑暗中,pp2cr突變植株中茉莉酸相關基因表現量較野生型低。綜合實驗結果推測,EGR2的作用時機為短暫照射藍光的情況下,影響FIN219/JAR1的磷酸化狀態,促進光反應、抑制茉莉酸反應;PP2CR則作用在持續黑暗或持續藍光等狀態,去磷酸化FIN219/JAR1,並促進茉莉酸反應。FIN219/JAR1在不同條件下,其磷酸化狀態會受到調控,並影響它在光和茉莉酸訊息傳遞中扮演的角色,最終影響阿拉伯芥幼苗的生長發育。 Photomorphogenesis in Arabidopsis is regulated by several hormones and photoreceptors. In order to respond to different environmental signals, post-translational modifications such as phosphorylation or dephosphorylation of proteins affect light signaling pathways. In previous studies, Casein Kinase 2 (CK2) could phosphorylate FIN219/JAR1, a jasmonate conjugating enzyme in Arabidopsis. By dark transition to blue light condition, phosphorylation status of FIN219/JAR1 has dynamic changes, which suggests that a protein phosphatase is involved in this process and different phosphorylation status FIN219 might affect light and jasmonate responses. By the use of Co-IP/LC-MS/MS assays and studies of blue light-regulated gene expression, Clade E Growth-Regulating Type 2C protein phosphatase2 (EGR2) and type 2C protein phosphatase related protein (PP2CR) were identified. EGR2 and PP2CR were further shown to have a physical interaction with FIN219 by Bimolecular Fluorescence Complementation (BiFC) assays in dark or blue light condition. Phos-tagTM acrylamide SDS-PAGE showed that the phosphorylation status and stability of FIN219 are affected by either EGR2 or PP2CR. The egr2 mutant showed insensitivity in blue light-mediated inhibition of hypocotyl elongation. Moreover, in MeJA-induced anthocyanin accumulation, egr2 had more anthocyanin accumulation as compared with Col-0, but pp2cr had less chlorophyll and anthocyanin accumulation in MeJA-induced chlorophyll and anthocyanin accumulation. Upon exposure to blue light 30 mins, the expressions of JA responsive genes were increased and light responsive genes decreased in egr2 compared with Col-0. In dark condition, JA responsive gene expression was decreased in pp2cr compared with Col-0. Taken together, under blue light condition, EGR2 regulates FIN219 phosphorylation status to promote light responses and inhibit JA responses; whereas, in continuous dark or blue light condition, PP2CR promotes JA responses by changing FIN219 phosphorylation status. Thus, under different conditions, phosphorylation status of FIN219 is regulated by different kinases and phosphatases. Phosphorylation status of FIN219 plays an important role in the crosstalk of light and jasmonate signaling to regulate Arabidopsis seedling development. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69387 |
DOI: | 10.6342/NTU201801415 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物科學研究所 |
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