探討阿拉伯芥中COP1與ERF1的交互作用以及光照與逆境交互影響下的生理功能

Abstract

因為氣候變遷的影響，極端的氣候變化正逐漸影響全球多個農業地區，其中對植物所造成了包含高鹽、乾旱、淹水等非生物逆境，使得農作物生長面臨挑戰，人類的糧食危機也正在醞釀。植物會因應不同逆境於轉錄層次進行基因調控，其中ETHYLENE RESPONSE FACTOR 1 (ERF1) 作為植物激素—乙烯，的下游基因，可調節生物和非生物逆境反應基因的表達，並在逆境耐受中發揮重要作用。根據前人的報導，ERF1 的蛋白在黑暗中很不穩定。在這項研究中，我們透過體外和體內的免疫沉澱分析證實了ERF1蛋白可以與 CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) 蛋白進行交互作用。COP1 是一種在光訊息傳遞中起負面調控作用的 E3 連接酶；我們利用雙分子螢光系統 (BiFC) 顯示了ERF1只能在黑暗下與 COP1 結合。為了瞭解COP1是否也參與在逆境反應下，我們進行了鹽逆境的測試。結果顯示，cop1 突變體相較於野生型有較長的主根以及較高的萌芽率，而且在cop1突變體中過度表達 ERF1 會提高它們的存活率；在黑暗條件下，ERF1下游基因，如P5CS1和PDF1.2，在cop1 突變體中的表達皆高於野生型，並在 ERF1 / cop1 植物中甚至更高，代表COP1 通過負調控ERF1表現來負調節部分的逆境反應。另一方面我們也好奇ERF1是否也參與了光訊號的傳遞；在下胚軸測試中，我們發現與野生型相比ERF1過表達植物的下胚軸較野生型的短，而ERF1 RNAi植株的下胚軸較長；在黑暗進入光照的條件下，與野生型相比ERF1過表達植株中的光反應相關基因，如Fd2、HEMA1、CAB3，都有顯著較高、葉綠素含量也明顯較高表現，代表ERF1在光型態發生中扮演正向調控者的角色。此外，COP1蛋白在黑暗條件下和ACC（乙烯前驅物）處理下會在細胞核中累積，代表除了光照以外尚有其他調節COP1的機制存在。我們的研究證實了一項調控光照與逆境交互影響下的分子機制。

ETHYLENE RESPONSE FACTOR 1 (ERF1) regulates biotic and abiotic stress-responsive gene expression and plays important role in stress tolerance. Previous report showed that ERF1 is unstable in the dark. Here, using in vitro and in vivo immunoprecipitation assays, we showed that ERF1 could interact with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), an E3 ligase that plays a negative role in light signaling. Moreover, bimolecular fluorescence complementation assay showed that ERF1 interacts with COP1 only in the dark condition. In our salt stress tests, cop1 mutants showed longer primary root elongation, and overexpressing ERF1 in cop1 mutants enhanced their survival rate. The expressions of ERF1 downstream genes, including P5CS1 and PDF1.2, were higher in cop1 mutants under darkness, and were even higher in ERF1/cop1 plants compared to WT, suggesting that COP1 negatively regulates stress responses through destabilizing ERF1. On the other hand, ERF1 overexpression plants showed shorter hypocotyl elongation whereas ERF1 RNAi knockdown lines showed longer hypocotyl elongation compared to WT. The light-responsive genes such as Fd2, HEMA1, CAB3 were significantly increased in ERF1 overexpression lines, suggesting that ERF1 plays a positive role in photomorphogenesis. In addition, COP1 protein accumulates in the nucleus in dark condition and under ethylene precursor treatment, indicating that there might be other mechanisms regulating COP1. Our results reveal a novel mechanism underlying the light-stress signaling crosstalk.