阿拉伯芥中FIN219/JAR1蛋白質磷酸化修飾在光與茉莉酸訊息傳遞中的功能性研究

Functional studies of FIN219/JAR1 phosphorylation involved in integration of light and jasmonate signaling in Arabidopsis

蔡易宏(Yi-Hung Tsai)

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(2016年)

https://doi.org/10.6342/NTU201602093

摘要

植物生長發育過程受數種荷爾蒙及多項環境因子如光等共同調控。幼苗發育早期，活化的遠紅光受體光敏素A(phytochrome A)、紅光受體光敏素B(phytochrome B)與藍光受體隱花色素(cryptochrome)皆參與荷爾蒙相關信息傳遞。近年研究指出FAR-RED INSENSITIVE 219 (FIN219)具催化茉莉酸(jasmonate)與異白胺酸(isoleucine)結合並生成具生物活性的茉莉酸外，更涉及藍光及遠紅光等傳訊路徑。實驗團隊先前亦發現受酪蛋白激酶(Casein kinase 2)所調控之FIN219蛋白質磷酸化將使其酵素活性改變。本研究嘗試透過FIN219蛋白質受酪蛋白激酶磷酸化位點突變的轉殖株，釐清其磷酸化狀態於植物生長發育早期所扮演的角色，並藉磷酸標籤蛋白質電泳，尋找可能利用FIN219為受質之磷酸化激酶。初步結果顯示，FIN219蛋白質兩個受酪蛋白激酶2磷酸化的胺基酸突變時，將造成FIN219蛋白質於藍光照射下穩定性改變，且此兩個胺基酸改變的雙突變體幼苗下胚軸長度於藍光下亦明顯低於野生型。另磷酸標籤蛋白電泳顯示，在黑暗、遠紅光與紅光中生長的野生型幼苗，含有磷酸化的FIN219蛋白質；相對的遠紅光及紅光照射下對野生型幼苗外源添加甲基茉莉酸，以及遠紅光下光敏素A突變體、黑暗中光敏素B突變體及藍光下酪蛋白激酶突變體皆造成幼苗累積較多未磷酸化的FIN219蛋白質。反之短暫藍光照射則可能藉隱花色素相關路徑，造成FIN219蛋白質去磷酸化。同時，體外磷酸激酶試驗亦指出光敏素A與光敏素B皆可造成FIN219蛋白質磷酸化，且未磷酸化之FIN219蛋白質呈現與PHYA及COP1蛋白質更高的親和力。綜合上述，光敏素及隱花色素皆可能透過調控FIN219蛋白質磷酸化狀態，以調控光型態發生的反應 。

關鍵字

FIN219 ； 磷酸化修飾 ； 茉莉酸 ； 光型態發生 ； 酪蛋白激酶

並列摘要

Several hormones and photoreceptors together regulate photomorphogenesis of seedlings. To respond to various environments, post-translational modifications such as kinase-mediated phosphorylation have been demonstrated to participate in several light signaling pathways. Previous studies revealed that FIN219/JAR1, a jasmonate conjugating enzyme, could be phosphorylated by casein kinase 2 (CK2). Enzyme kinetic analysis also showed that unphosphorylated FIN219 exhibited higher substrate affinity. To further determine the roles of two phosphorylation isoforms of FIN219, transgenic lines containing FIN219 native promoter driven substitution of residues that were demonstrated to be phosphorylated by CK2 showed the phenotypic effects on methyl jasmonate (MeJA)-mediated inhibition of hypocotyl elongation and increased accumulations of dephosphorylated FIN219 under blue light. On the other hand, Phos-tagTM acrylamide SDS-PAGE electrophoresis showed that most of FIN219 would be phosphorylated in the dark, red and far-red light, but exogenous MeJA could trigger dephosphorylation of FIN219 under far-red light and red light. Additionally, kinase assays indicated that PHYA and PHYB could phosphorylate FIN219 in vitro, which is consistent with the fact that only the unphosphorylated form of FIN219 exists in both phyA null mutant under far-red light and phyB null mutant in the dark. Protein interaction assays also revealed that dephosphorylated FIN219 exhibited higher affinity
to PHYA and COP1. Interestingly, blue light irradiation resulted in dephosphorylation of FIN219 in a cryptochromes-dependent manner. Taken together, current data indicate that photoreceptor-mediated phosphorylation changes of FIN219 plays a vital role in regulating photomorphogenic development of Arabidopsis seedlings.

並列關鍵字

FIN219 ； phosphorylation ； jasmonate ； photomorphogenesis ； casein kinase 2

參考文獻

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