| The growth,development,yield and quality of citrus are seriously affected by drought and high salinity.It is an important means to create stress resistance germplasm by using the transcription factor with diverse function.It also provides a new way of thinking for the citrus plant molecular improvement.WRKY is a family of plant specific transcription factor,which plays an important role in plant response to biotic and abiotic stresses.Although some WRKY transcription factors have been identified in different plants,the function and regulatory network of most WRKYs remain unclear.In our earlier study,the role of a kumquat transcription factor FcWRKY40 in antioxidant stress has been analyzed,this study is aimed in analyze the function of FcWRKY40 in response to abiotic stress(salt and drought)and elucidate its functional mechanism.The main results are as follows:1.Functional evaluation of salt resistance of FcWRKY40.The tobacco transgenic lines with overexpression of FcWRKY40 showed higher germination rate than WTt under salt stress.The growth performance was better than WTt,concurrent with significantly lower EL,MDA and ROS content,the chlorophyll content and photosynthetic efficiency were higher in comparison with WTt,meanwhile,transgenic lemon plants overexpressing FcWRKY40 were obtained by Agrobacterium-mediated transformation.Under salt stress,transgenic lines of lemon were influenced to a less serious extent than that of WTL,while the chlorophyll content was higher than WTL and the ROS accumulation was lower than WTL.Virus-induced gene silencing(VIGS)was used to obtain FcWRKY40-silencing kumquat.The VIGS lines was more seriously injured under salt stress,in consistent with reduced chlorophyll content,and accumulation of more ROS,indicating that FcWRKY40 plays a positive role in salt resistance.2.FcWRKY40 binds and activates the promoters of FcSOS2 and FcP5CS1.The expression of nine genes associated with salt stress in lemon transgenic line was analyzed,which demonstrated that three genes of SOS pathway,SOS1,SOS2 and SOS3,and P5CS1,which is the key enzyme of proline synthesis,were up-regulated in the transgenic plants relative to WTL.In contrast,their transcript levels were down-regulated in the VIGS line.Physiological tests showed that Na~+content in the lemon transgenic plants was decreased,while K~+content and proline was increased.By contrast,VIGS lines showed an opposite trend of ion content and proline accumulation.Treatment of P5CS1 synthetic inhibitor could compromise salt tolerance of the transgenic lemon,while supplementation of exogenous proline partially recovered the salt tolerance of the VIGS lines.The promoters of Kumquat FcSOS1,FcSOS2,FcSOS3 and FcP5CS1 contain two,one,four and two W-box elements,respectively.Yeast one hybrid assay,LUC and EMSA confirmed that FcWRKY40 specifically interacted with the W-box elements on FcSOS2 and FcP5CS1 promoters and activated their expression.In addition,FcWRKY40 could be induced by salt and ABA,and salt-induced expression depends on ABA synthesis.The FcWRKY40 promoter has ABF transcription factor binding element ABRE,and yeast one hybrid assay,LUC and EMSA indicated that FcWRKY40 could be directly regulated by FcABF2 and acts as the target gene of the latter.3.Functional evaluation of FcWRKY40 in drought resistance.Overexpression of FcWRKY40 in tobacco showed greater resistance to dehydration and drought stress.The relative water loss,MDA,EL and ROS levels were significantly lower than those in WTt,while chlorophyll content was significantly higher than that of WTt.The transgenic lemon showed less serious leaf wilting than WTL after dehydration,accompanied by lower relative water loss,MDA and ROS contents.On the contrary,an manifested by silencing of FcWRKY40 led to enhanced drought susceptibility,increased EL,MDA and ROS,and reduced chlorophyll content was decreased.All these findings indicated that FcWRKY40 plays a positive role in the drought resistance of plants. |
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