Functional Analysis Of Transcription Factor ZmbZIP4 In Maize

Maize(Zea mays L)is one of the most important crops for livestock and humans.Its growth and development are frequently affected by various abiotic stresses,such as high salinity,drought,heat,and low temperature,which negatively affect crop growth and productivity.Therefore,it is imperative to elucidate the mechanisms of stress responses in maize.Many previous studies have demonstrated when the plant is stimulated by environment factors it will transmit the signal into the nucleus through a series of events for signal transduction,and regulate the expression of related genes by transcription factors.The phytohormone abscisic acid(ABA)plays a critical role in plant stress response and regulates the expression of many stress response genes involved in stress response and tolerance.From the promoters of those stress-responsive genes,a major cis-acting element(ABRE)was characterized as being necessary for their response to ABA.When plants suffer from abiotic stress,the content of ABA increased,and activated SnRK2s protein kinases phosphoiylate some transcription factors,which regulate the expression of stress-related genes.In the analysis of transcriptome data from the primary root and ovary of maize under drought stress conditions,a bZIP(basic leucine zipper)transcription factor was found and named as ZmbZIP4,which was induced to up-regulated expression by drought stress.In the pattern plants,bZIP transcription factors regulate diverse processes such as development and stress responses.However,few of them have been functionally characterized in maize.ZmbZIP4 is a stress-responsive transcription factorThe ZmbZIP4 is an orthologue of AREB1(ABA-RESPONSIVE ELEMENT BINDING PROTEIN1)in Arabidopsis and OsbZIP23 in rice from the phylogenetic relationship of the group A bZIP proteins based on the amino acid sequences of these proteins.The expression of ZmbZIP4 in maize seedlings and mature plants is ubiquitous,but the abundances of ZmbZIP4 transcripts show differences across different organs.The expression of ZmbZIP4 is remarkably induced by high salinity,polyethylene glycol(PEG)treatment,oxidative stress(H2O2),heat stress and phytohormones ABA or indole-3-acetic acid(IAA)treatment to up-regulated strongly,and only slightly affected by cold,gibberellic acid(GA)and jasmonic acid(JA)treatment.And the transactivation assay in yeast demonstrated that ZmbZIP4 function as a transcriptional activator to response to abiotic tresses.Overexpression of ZmbZIP4 in maize improves the root development and resistance to abiotic stresses.ZmbZIP4 was transformed into the maize inbred line DH4866 to produce overexpressed lines,and two zmbzip4 UFMu homozygous mutant lines with different insertion sites were selected from the mutants of Maize Genetics Cooperation Stock Center by self-fertilization and molecular identification.The performances of the ZmbZIP4 overexpressed lines,the zmbzip4 mutants contrast of their WT controls were identified.The ZmbZIP4 overexpressed lines had a developed root system and improved salt and drought resistance,and the mutants were opposite.The root number,root length and lateral-root primordia were counted on maize plants cultured in nutrient solutions,and the data showed that the ZmbZIP4 overexpressed lines had longer primary roots,seminal roots,and lateral roots compared with the WT(DH4866).The differences of root system and biomass between the transgenic lines and WT were principally caused by the number and length of lateral roots.And the results of the Feulgen staining of the lateral-root primordia(LRP)also showed that the LRP number in the ZmbZIP4 overexpressed lines was significantly more than the WT.When plants both in solution culture and in soil cultivation grew in high salt or drought(osmotic stress)environment,the ZmbZIP4 overexpressed plants had significantly higher stress resistance than that of control,while zmbzip4 mutants showed significantly reduced stress resistance.The contents of ABA in the ZmbZIP4 overexpressed lines,the mutants and the controls were determined under normal and salt stress conditions.And the ZmbZIP4 overexpressed plants accumulated more ABA,while in the mutants ABA contents were decreased.It could be concluded that the overexpression of ZmbZIP4 promoted the accumulation of ABA in maize and the improvement of plant stress resistance and root development.ZmbZIP4 regulates the expression of some stress response and ABA synthesis genesTo investigate the function of ZmbZIP4 in stress resistance and plant development,maize transgenic lines with ZmbZIP4-GFP structure were produced by genetic transformation.The downstream target genes of ZmbZIP4 were identified by ChIP-Seq with ZmbZIP4-GFP transgenic maize plants at the three-leaf stage and the monoclonal antibody of GFP.A number of genes involved in response to salt and drought stress,including ZmRD20,ZmRD21,ZmNHX3,ZmGEA6,ZmNF-YA4 and ZmLEA2,were identified by the analysis of the ChIP-Seq data,ChIP-qPCR,RT-PCR and the transactivation assay in the yeast.In addition,some genes participated in ABA synthesis were identified,including ZEP/ABA1,NCED,AA03 and LOS5/ABA3,among them NCED was the rate-limiting gene in ABA synthesis.The promoter element analysis and EMSA detection showed these genes were the candidate target genes of ZmbZIP4,and the cis-acting element "ACGTG" in their promoter could be specifically bound by ZmbZIP4.They were positively regulated to up-expression by ZmbZIP4.The higher expression of the genes participated in ABA synthesis led to higher levels of ABA in ZmbZIP4-overexpressing lines.Multiple root development related genes are targeted by ZmbZIP4Some genes involved in maize root development,including ZmSCR,ZmLRP1,ZmIAA8,ZmIAA14,ZmARF2 and ZmARF3,as candidate target genes for ZmbZIP4 were identified by ChIP-Seq,ChIP-qPCR,yeast single hybridization and Luciferase activity assay.The orthologues of these genes in the model plants participate principally in the regulation of root development,especially in the process of lateral root formation.ZmSCR1.ZmLRPl,ZMARF2,and ZmARF3 were up-regulated in the ZmbZIP4 overexpressed lines compared to the WT,while ZmIAA8 and ZmIAA14 were down-regulated,both of them are transcriptional repressors for auxin response genes.In the zmbzip4 mutants,the expression levels of these genes showed opposite changes ZmbZIP4 regulated the root development of maize by regulating the expression of some genes involved in root development.ZmbZIP4 regulated the expression of the genes via ABA signaling pathwayUsing ZmbZIP4 as bait for yeast two-hybrid screening,a number of proteins interacted with ZmbZIP4 were selected.These proteins function mainly in stress response and glucose metabolism.Combined with the nuclear localization features of ZmbZIP4 and its biological functions in maize,four proteins that might interact with ZmbZIP4 were selected for Co-IP validation.The results showed ZmbZIP4 could interact with ZmPYL12,ZmMAPK17,ZmEREB182 and ZmNTF1.ZmPYL12 is an ABA receptor protein,which suggests that ZmbZIP4 may be involved in ABA signaling pathway.By using yeast two-hybrid assay to verify the interaction between members of ZmSnRK2s and ZmbZIP4,the results showed that ZmbZIP4 could interact with SnRK2.1,SnRK2.3,SnRK2.6,SnRK2.10,SnRK2.11.In the ZmbZIP4 overexpressed lines the expression of ZmSnRK2.1,ZmSnRK2.3 and ZmSnRK2.10 was up-regulated.Based on the reports that ZmSnRK2.1,ZmSnRK2.3,ZmSnRK2.10 can interact with ZmPP2Cl or ZmPP2C6,which implies that ZmbZIP4 is involved in ABA signaling pathway.It is concluded ZmbZIP4 is an important partner in the formation of oligomers of ZmbZIP4 with ZmSnRKs,and/or with ZmPP2Cs in maize.It could be inferred that the phosphorylation state of ZmbZIP4 may impact on its function,ABA signal transduction and amplification.