Functional Characterization Of The BpbHLH112 Gene In Modulating Salt And Drought Tolerance In Betula Platyphylla

The basic/helix-loop-helix（bHLH）transcription factor family exists in all three eukaryotic kingdoms.Plant bHLH family members are numerous and involved in complex biological processes.Several studies have reported that bHLHs play an important role in the growth,development,and stress responses in plants.In previous study,a bHLH transcription factor,designated BpbHLH112,was found to respond to salt stress through comparative transcriptome analysis in birch.In present study,the expression patterns and the role of BpbHLH112 gene in salt and drought stress response were investigated in Arabidopsis thaliana and Betula platyphylla Suk,respectively.The results are as follows:（1）The BpbHLH112 gene was cloned from B.platyphylla Suk.The BpbHLH112 coding sequencing（CDS）is 1431bp long,encodes 476 amino acids residues and has a molecular mass of～52 k D and an isoelectric point of 5.78.The prediction of conserved domains and phylogenetic analysis revealed that BpbHLH112 belongs to the bHLH transcription factor family.（2）The expression pattern of BpbHLH112 was analyzed.Expression profile analysis showed that BpbHLH112 was differentially expressed in various organ including roots,stems,leaves and bud tissues.Besides,the expression of BpbHLH112 in seedlings could be induced under salinity,drought or ABA treatment suggesting that BpbHLH112 was involved in abiotic stress response in B.platyphylla.（3）The BpbHLH112 was fused with a GFP reporter gene and transient expression of BpbHLH112-GFP in tobacco epidermal cells revealed that BpbHLH112 was a nuclear-localized protein,indicating that BpbHLH112 functions in the nucleus as a transcription factor.Furthmore,transactivation assay demonstrated that BpbHLH112 possesses transactivation activities at its N-terminus in yeast.（4）The 1446 bp promoter region of BpbHLH112 from birch leaves was cloned.Bioinformatic analysis revealed that it contains numerous cis-acting elements associate with stress responsiveness and plant hormones motifs.The BpbHLH112 promoter was further fused with a GUS gene and introduced into birch plantlet.Transient expression of BpbHLH112pro-GUS in birch showed that BpbHLH112 promoter could drive GUS expression in roots,stems,leaves and buds of plantlet,consistent with its expression pattern.In addition,four 5’-deletion fragments obtained from the BpbHLH112 promoter were fused with a GUS reporter gene and used for tobacco transient assay.The results of GUS histochemical staining and GUS activity assay showed that the salt stress positive regulatory region of the BpbHLH112 promoter was-347 to+55,the positive regulatory region of drought stress is-1446 to-1031,and the positive regulatory region of ABA stress is-634 to-347,which proves that the BpbHLH112 gene promoter can regulate a variety of adversity stresses and is a stress-inducible promoter.（5）To elucidate the potential biological functions of BpbHLH112,transgenic A.thaliana overexpressing BpbHLH112 were generated.Eleven transgenic lines were generated verified.Under stressed conditions,Arabidopsis lines over-expressing BpbHLH112 had superior phenotype and the germination rates were higher than those of the wild-type plants,as were the root lengths.Additionally,the BpbHLH112-overexpression lines had lower malondialdehyde（MDA）and H₂O₂contents,but higher peroxidase（POD）and superoxide dismutase（SOD）activities as well as higher proline content in comparison to the wild-type plants.These results suggest that in transgenic Arabidopsis over-expression of BpbHLH112 enhances tolerance to drought and salinity stress.（6）To further verify the role of BpbHLH112 in response to drought and salt stress,the BpbHLH112 gene was overexpressed in B.platyphylla.The transgenic plants exhibited superior phenotype,an increased chlorophyll and proline content as well as the activities of SOD and POD,but decreased MDA content compared with wild-type plants.Furthermore,the transgenic plants displayed an enhanced Reactive oxygen species（ROS）scavenging ability,which resulted in an efficient maintenance of oxidant–antioxidant homeostasis,indicating that overexpression of BpbHLH112 confers enhanced tolerance to drought and salinity stress in transgenic birch.Collectively,these results suggest that BpbHLH112 functions as a bHLH transcription factor which plays positive roles in salt and drought tolerance in both Arabidopsis and birch seedlings,indicating that it can potentially be used in transgenic technology approaches to improve birth drought and salt tolerance.