阿拉伯芥中透過去磷酸酶調節FIN219/JAR1活性並影響藍光及茉莉酸訊息傳遞之功能性研究

Functional studies of Arabidopsis phosphatases in regulating FIN219/JAR1 activity upon exposure to blue light and jasmonates

朱彥樺(Yen-Hua Chu)

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(2020年)

https://doi.org/10.6342/NTU202100139

摘要

阿拉伯芥幼苗透由光受體接收光訊號後,會活化多種具酵素活性的蛋白質並協同賀爾蒙調控光型態發生，並透過轉錄因子或關鍵蛋白質的後轉譯修飾像磷酸化改變狀態，來針對周圍環境做出反應。先前的研究中，藍光環境下， 酪蛋白激酶2(CK2) 會磷酸化FIN219/JAR1。 FIN219/JAR1催化茉莉酸(jasmonate)和異白胺酸(isoleucine)結合，以活化茉莉酸訊息傳遞， 並同時促進植物的光型態發生。 利用LC-MS/MS及microarray分析得到可能參與在藍光中FIN219/JAR1去磷酸化功能之去磷酸酶: 蛋白質去磷酸酶2C型E支的EGR2和H支的PP2CR， 以及蛋白質去磷酸酶2A次單位A 的RCN1蛋白質。目前為止，FIN219不同磷酸化形式的功能仍是未知。在此研究中，使用上述得到之去磷酸酶突變的植株去闡述FIN219/JAR以何種形式來應對藍光及茉莉酸訊息傳遞。利用雙分子螢光互補實驗(BiFC)及in vitro pull-down分析得知FIN219與這些去磷酸酶有直接的交互作用關係。萃取去磷酸酶突變株植物(egr2，pp2cr，rcn1) 蛋白質，並使用磷酸標籤蛋白質電泳，確認egr2，pp2cr照射藍光及外加MeJA處理下，FIN219蛋白質穩定度與磷酸化的形式增加，而rcn1中FIN219磷酸化是不受光調控的。另外植物外加MeJA時，造成egr2，pp2cr和rcn1下胚軸生長抑制，發芽率受到影響, 花青素累積下降以及側根優勢生長，都顯示外加MeJA會使EGR2， PP2CR和PP2ARCN1持續去磷酸化FIN219，以抑制過度的茉莉酸誘導活化。相較於專一性藍光調控的EGR2和 PP2CR，PP2ARCN1則是獨立於光的調控。總體而言，阿拉伯芥的幼苗發育中，FIN219不同的磷酸化形式在串聯藍光及茉莉酸訊息傳遞路徑當中扮演重要的角色。

關鍵字

FIN219 ； 藍光型態發生 ； 茉莉酸訊息 ； 去磷酸酶 ； 磷酸化

並列摘要

Arabidopsis seedlings activate multiple enzymatic proteins and coordinate hormones while receiving light signals via photoreceptors to facilitate photomorphogenesis. In order to adapt environments, key transcription factors or enzymes in plants undergo post-translational modifications such as phosphorylation. Previous studies indicated that Casein Kinase2 (CK2) phosphorylated FAR-RED INSENSITIVE 219/JASMONATE RESISTANT1 (FIN219/JAR1) under blue light. FIN219/JAR1 is a jasmonate conjugating enzyme and controls photomorphogenesis in Arabidopsis. Several phosphatases identified by LC-MS/MS and microarray analysis may regulate FIN219 under blue light: the clade E Type 2C protein phosphatase 2 (EGR2), the clade H type 2C protein phosphatase (PP2CR), and phosphatase 2A regulatory subunit A (RCN1). So far, the functions underlying phosphorylation and dephosphorylation of FIN219 remain unknown. In this study, we use these phosphatases to elucidate the functions of FIN219 dephosphorylation in response to blue light and JA signaling pathway. Bimolecular Fluorescence Complementation (BiFC) and in vitro pull-down assays demonstrated interaction between FIN219 and these phosphatases. Phos-tag acrylamide SDS-PAGE studies showed that exogenous MeJA treatment increased the stability of phosphorylated FIN219 in egr2 and pp2cr mutants under blue light condition, while phosphorylated FIN219 accumulation in rcn1 is independent of light. Moreover, the phenotypic responses, including inhibition of hypocotyl elongation, anthocyanin accumulation, a decrease of germination rate, and increased lengths of lateral roots indicated that EGE2, PP2CR and PP2ARCN1 functionally suppressed excessive JA responses with MeJA treatment since EGR2 and PP2CR functionally depend on blue light, but PP2ARCN1 is independent of light. Taken together, the phosphorylation status of FIN219 plays an important role in the crosstalk of blue-light and JA signaling pathway to regulate Arabidopsis seedling development.

並列關鍵字

FIN219 ； blue-light photomorphogenesis ； Jasmonate signaling pathway ； phosphatase ； phosphorylation

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