Two BHLH Homologous Genes Negatively Regulate ABA-dependent Drought Response In Arabidopsis Thaliana And Soybean

Abscisic acid(ABA)participates in plant responses to environmental stresses such as drought,high salinity and cold,and these responses are precisely regulated by transcription factors.There are few reports regarding ABA-dependent drought response mediated by bHLH transcription factors,especially in soybean.GmbHLH 120.a drought-ielated transcription factor in soybean,has been identified in laboratory before.Its homolog in Arabidopsis thaliana is AtbHLH018.Based on researches of AthHLH018 in Arabidopsis thaliana,we found that two transcription factors are involved in ABA-dependent drought response through applying a series of phenotypic and functional analysis.Our detailed results are listed as follows:1)The AtbHLH018 from is an homologous gene far GmbHLH120 from soybean.Both of them contain conservative amino acids of bHLH domain.There is 38.9%sequence similarity of two proteins,and they are derived from the same node of phytogenic tree,indicating that the two typical bHLH transcription factors are homologous genes between Arabidopsis and soybean.2)We found that AtbHLH018 gene can negatively regulate drought response in Arabidopsis via ABA-dependent stomata closure.These results shown that the expression level of AtbHLH018 is significantly reduced after either exogenous PEG treatment or ABA treatment.atbhlh018 mutants are mare insenative to PEG treatment compared to wild-type he water loss rate of detached leaves from atbhlh018 mutants were reduced.and stomatal apertures of atbhlh018 mutants were decreased.The dual luciferase Teporter assay proved that AtbHLH018 has transcriptional activation activity.Both germination and seedling growth of atbhlh018 mutants were more sensitive to ABA treatment.3)We also found that GmbHLH120 gene can negatively regulate drought response in soybean via ABA-dependent stomata closure.For creating gmbhlh120 knockout mutants though CRISPR/Cas9 system.transgenic plants containing CRISPR knockout vectors were produced by soybean genetic transformation.For gmbhlh120 mutants,two independent homozygous mutated lines were achieved.The water loss rate of gmbhlh120 mutants detached leaves were reduced,and stomatal apertures of gmbhlh120 mutants were decreased.GmbHLH120 overexpressing transgenic hairy roots exhibited enhanced PEG sensitivity and reduced ABA sensitivity.Above all,two homologous genes including AtbHLH018 and GmbHLH120 negatively regulate drought response in Arabidopsis thalian and soybean,respectivly.Moreover,this regulation relies on the AB A-dependent stomata closure,which builds a research basis for exploring the molecular mechanism of plant responses to drought mediated by b-HLH gene.