The Mechanism Of Arabidopsis Trihelix Transcription Factor AST1 In Drought Or Salt Stress Response Regulation

Trihelix transcription factors are characterized by containing a conserved trihelix(helix-loop-helix-loop-helix)domain that bind to GT elements required for light response,play roles in light stress,and also in abiotic stress responses.However,only few of them have been functionally characterised.In the present study,we characterized the function of AST1(Arabidopsis SIP1 clade Trihelixl,AT3G24860)in response to abiotic stress.(1)The Fusion expression vector PBI121-ProAST1-GUS was successfully constructed and transformed into Arabidopsis thaliana using the floral dip method.GUS staining was performed that ASTI was expressed at each studied developmental stage and in different tissues.The expression of ASTI increased along with the growth of plants,but reduced when plants began to stem elongation.GUS staining and qRT-PCR showed that ASTI is highly expressed in stems,leaves and flowers,but has relative lower expression levels in roots and siliques.qRT-PCR showed that AST1 could response to salt and osmotic stresses.Consistently,determination of.GUS activity in A.thaliana plant expressing ProASTl:GUS also confirmed that the expression of AST1 was significantly induced in leaves and roots after exposed to mannitol or NaCl.The Fusion expression vector PBI121-ProAST1-GFP was successfully constructed,and the GFP signal was detected in the whole cells of root tips or root elongation zone in plants expressing 35S:GFP,and the GFP signal was only detected in the nucleus of the root tips to the root hair zone in plants expressing AST1-GFP.Additionally,transient transformation of onion epidermal cells also indicated that ASTI is localized in the nucleus.(2)The overexpression vector pROK2-AST1 was successfully constructed and transformed into A.thaliana using the floral dip method.The T3 generation of A.thaliana plants overexpressing ASTI(OE)and the AST1 hojmozygous mutant plants(KO plants)were generated.Compared with WT and WT plants transformed with the empty pROK2 vector(35S),ASTI can regulate salt and osmotic stress tolerance positively.By the analysis of the physiological response mediated by AST1 under NaCl and Mannitol conditions,it was found that ASTI controls stomatal closure and opening by regulating water stress tolerance,Maintenance of K+/Na+ homeostasis,proline biosynthesis,reduce membrane lipid peroxidation,and increase reactive oxygen species scavenging capability.qRT-PCR analysis showed that AST1 could regulate the expressions of AtMYB61,Na+(K+)/H+ transport proteins,proline biosynthesis and degradation genes,SODs,PODs,and LEA family genes to improve the stress resistance.(3)A transcriptomic analysis was carried out to identify differentially expressed genes(DEGs)between the OE3 and KO1.1.2 lines.In total,144 DEGs(fold change>2 and false discovery rate(FDR)<0.05)were identified,among which 65 genes were upregulated and 77 genes were downregulated.Gene ontology(GO)analysis showed that these DEGs are mainly involved in signaling,immune system process,reproduction and cell killing.The promoters of genes that were highly upregulated by ASTI according to the qRT-PCR and RNA-Seq analyses were used for MEME analysis,and the results showed that there was a 12 base conserved sequence present in most of the studied promoters.By Y1H,GUS assay,EMSA(Electrophoretic Mobility Shift Assay)andChIP(Chromatin immunoprecipitation),the core sequence which is bound by AST1 was termed AGAG-box with the sequence[A/G][G/A][A/T]GAGAG.Meanwhile,by Y1H,GUS assay,EMSA(Electrophoretic Mobility Shift Assay)and ChIP(Chromatin immunoprecipitation),AST1 also binds to some GT motifs including GGTAATT,TACAGT,GGTAAAT and GGTAAA,but failed in binding to GTTAC and GGTTAA.Chromatin immunoprecipitation combined with qRT-PCR analysis suggested that ASTI binds to AGAG-box and/or some GT motifs to regulate the expression of stress tolerance genes.In conclution,AST1 binds to AGAG-box and/or some GT motifs to regulate the expression of stress tolerance genes,resulting in reduced reactive oxygen species,Na+ accumulation,stomatal apertures,lipid peroxidation,cell death and water loss rate,and increased proline content and reactive oxygen species scavenging capability.These physiological changes mediated by AST1 finally improve abiotic stress tolerance.