Functional Characterization Of CsCBF1 From Sweet Orange(Citrus Sinensis)in Regulation Of ADC Gene Expression And Putrescine Biosynthesis

Citrus is an important fruit tree in China,and sweet orange?Citrus sinensis?accounts for more than half of the total production of citrus industry.Low temperature is a limiting environmental factor for sweet orange production.Genetic engineering is a feasible means to improve cold resistance,the premise is to have key genes for cold resistance.The complete sequencing of sweet orange gene is important for elucidating the mechanism of low temperature response of sweet orange,excavating and identifying cold resistance genes.It has been shown that polyamines play critical roles in plant responses to stresses.ADC?arginine decarboxylase?is an important rate-limiting enzyme in the polyamine synthesis pathway.CBFs?C-repeat binding factors?are important regulators implicated in low temperature signal transduction pathway of plants,but it remains unclear whether CBFs can regulate ADC gene expression and polyamine biosynthesis in response to low temperature in sweet orange.The present work aims to investigate CBF regulates ADC gene expression and putrescine accumulation under low temperature conditions.The main results are as follows:1.CsCBFs of sweet orange were responsive to a variety of abiotic stresses,such as low temperature,salt,ABA,dehydration,in which CBF1 was most obviously induced by low temperature.Transgenic sweet orange lines overexpressing CsCBF1were obtained by Agrobacterium-mediated genetic transformation,while VIGS?virus-induced gene silencing?was employed to knock down PtrCBF1?a CsCBF1homologe gene?in Poncirus trifoliata.2.Cold tolerance of the overexpressing lines and VIGS plants was assessed.the overexpressing lines accumulated less hydrogen peroxide and exhibited greater antioxidant enzyme activity and higher ADC expression level,along with increased endogenous putrescine content,in comparison with the wild type?WT?under cold treatment.By contrast,the VIGS lines displayed enhanced cold sensitivity than the TRV control,concurrent with more ROS but less putrescine content.3.The VIGS plants pretreated with exogenous putrescine and the transgenic plants were treated with D-arginine,an ADC inhibitor,prior to cold treatment.Exogenous supply of putrescine to the VIGS plants increased endogenous putrescine level,and the VIGS plants accumulated less H₂O₂,higher antioxidant enzyme activit in comparison with the water treatment after freezing treatment,indicating that putrescine can resume cold tolerance of the VIGS plants.On the other hand,exogenous application of D-arginine decreased endogenous putrescine content.Both overexpressing and wild-type lines exhibited wilting under cold stress.The D-arginine-treated plants exhibited increased cold susceptibility when compared to the control.4.Electrophoretic mobility shift assay?EMSA?showed that CsCBF1 could bind specifically to the CCGAC element on the ADC promoter.Taken together,these results demonstrated that CsCBF1 could regulated the expression of ADC gene,by binding to CRT/ERF element within the ADC promoter,and putrescine synthesis,which plays a role in cold response.