Genome-wide Identification Of BHLH Transcription Factor Family In Sweet Orange(Citrus Sinensis) And Functional Characterization And Mechanism Analysis Of CsbHLH18 In Cold Resistance

The basic helix-loop-helix(b HLH)transcription factors(TFs)comprise one of the largest gene families in plants.They not only widely participate in various physiological processes,but also play key roles in response to various biotic and abiotic stresses.Despite the characterization of b HLH genes in various plants,physiological role and regulatory function of the majority of b HLHs remain poorly understood.In this study,based on the sweet orange datebase information,we first performed a bioinformatics analysis of Csb HLH genes at genome-wide level,then screened candidate genes that were induced by cold stress treatment.Physiological and biochemical experiments were carried out to analyze the function of one of these candidate genes under cold stress and its potential mechanism.The main results are as bellow.1.A total of 56 putative Csb HLH genes were identified in sweet orange(Citrus sinensis)based on a genome-wide analysis.The Csb HLHs,except four members,were distributed throughout nine chromosomes and divided into 19 subgroups.Members of the same subfamily share similar gene structure and DNA binding activity.2.Most of the Csb HLH genes were expressed with divergent expression patterns upon cold exposure,with the greatest transcriptional upregulation being observed in Csb HLH18.The CDs of Csb HLH18 is 783 bp,which encodes a b HLH protein of 260 amino acids.The calculated molecular mass and theoretical isoelectric point of Csb HLH18 are 28.92 k Da and 7.09,respectively.Csb HLH18 is localized in the nuclei and has transcriptional activation activity which displayed in N terminal.3.Over-expression of Csb HLH18 conferred enhanced cold tolerance in transgenic tobacco,as manifested by higher survival rate,lower electrolyte leakage,better photosynthesis capacity and higher levels of proline in comparison with the wild type(WT).Similarly,transgenic lemon overexpressing Csb HLH18 displayed lower electrolyte leakage and MDA content after cold stress.In addition,the transgenic plants accumulated significantly less reactive oxygen species(ROS),concurrent with increased activities and transcript levels of antioxidant enzymes.By contrast,knockdown of b HLH18 gene by RNA interference in trifoliate orange promoted cold susceptibility,accompanied by down-regulation of antioxidant genes and accumulation of more ROS.4.Yeast one-hybrid assay,EMSA and dual luciferase assay demonstrate that Csb HLH18 could directly and specifically bind to and activate the promoter of Cs POD.On the other hand,Cs ICE1 was shown to interact with Csb HLH18 and regulates Cs SOD and Cs CAT.Taken together,these findings indicate that Csb HLH18 plays a positive role in cold tolerance through,at least partly,modulation of ROS homeostasis by directly regulating the antioxidant gene.5.RNA-Seq was performed using wild type and over-expression lines of lemon plants before and after cold treatment.We found that the number of differential genes enriched in plant hormone signaling pathway was the most abundant and showed most significant enrichment degree.There were also some differentially genes enriched in the starch and sucrose synthesis pathway,suggesting that the mechanism by which Csb HLH18 enhances cold resistance in plants may be related to these pathways,but the specific mode of action needs further experimental verification.