Studies On The Mechanism Of AtERF74 Responsed To Abiotic Stress In Arabidopsis Thaliana

As a result of global warming,multiple environmental stresses contribute towards limitations in plant and crop growth.Recent studies indicate that the ethylene response factor VII?ERF-VII?transcription factor is an important regulator of osmotic and hypoxic stress responses in plants.However,the molecular mechanism of ERF-VII-mediated transcriptional regulation remains unclear.Here,we investigated the role of AtERF74?a member of the ERF-VII protein family?by examining the abiotic stress tolerance of an ERF74 overexpression line and a T-DNA insertion mutant using flow cytometry,qPCR,transactivation and electrophoretic mobility shift assays.?1?It was found that in 25 out of 44 independent transgenic plants with ERF74repression?ERF74-DR?,the leaves were darker in color with over 100-fold increase in anthocyanins compared to the wild-type?WT?when grown under white light with an intensity of 100?mol·m^-2·s^-1.When 18-day-old ERF74-DR plants were grown under drought conditions for 15 days,ERF74-DR showed severe wilting symptoms compared with WT plants.?2?ERF74 and ERF75 respond strongly abiotc stresses such as flooding,drought,high light,heat and AlCl₃,whereas ERF71,ERF72 and ERF73 only respond to a subset of the stresses.?3?Transgenic plants overexpressing ERF74 showed enhanced tolerance to different abiotic stresses,whereas its T-DNA insertion mutant erf74 and erf74erf75 double mutant displayed higher sensitivity.?4?Subcellular localization results showed that under normal growth conditions,only23%of the cells exhibited a GFP signal in the nucleus with most of the GFP signal being membrane localized.In contrast,flooding,drought and HL conditions resulted in 92%,65%and 86%of the cells exhibiting a GFP signal in the nucleus.These results show that expression of ERF74 and ERF75 was induced and the protein translocated from the membrane to the nucleus upon different abiotic stresses.?5?The accumulation or scavenging of intracellular ROS in 35S::ERF74,WT,erf74,erf75,erf74erf75 protoplasts upon different stresses was measured by FCM with H₂DCFDA?2',7'-dichlorodihydrofluorescein diacetate?staining.And the results indicate that the production of ROS in the early stages of the response to the tested stresses may be associated with ERF74.?6?The levels of the ROS production genes including RbohD,RbohF,PRX33 and PRX34 were measured under different stress conditions in 35S::ERF74,WT,erf74,erf75,erf74erf75 plants using qRT-PCR.In the early stages of flooding,drought or HL condition,35S::ERF74 plants exhibited a pronounced increase in the expression of RbohD,whereas erf74 and erf74erf75 lines showed a smaller increase at the same time point.?7?Transactivation and electrophoretic mobility shift assays showed that ERF74 binds to the promoter of RbohD through GCC box.?8?Induction of stress marker genes and genes encoding ROS scavengers under various stress conditions are dependent on RbohD expression and ROS production.Above all,we propose a pathway involving ERF74 acting as on-off switch controlling an RbohD dependent mechanism in response to different stresses and maintaining H₂O₂ homeostasis in Arabidopsis.