Preliminary Study on Molecular Mechanism of ZmMKK3 in Response to High Temperature Stress

LIU Huafeng; YE Feiyu; ZHANG Dongling; TIAN Yanyu; WANG Guorui; MA Chenchen; LU Zihan; ZHANG Xin; CAO Liru; LI Huiqun

doi:10.15933/j.cnki.1004-3268.2026.02.003

Abstract：Previously,the specifically differentially expressed gene ZmMKK3 responding to high-temperature stress in heat-tolerant maize cultivar Zhengdan 819 was identified from transcriptome data of Zhengdan 819 and its parental lines before and after high temperature stress.Building on this,bioinformatics methods were employed to predict the structural domains,conserved motifs,and secondary structure of ZmMKK3 protein.Maize protoplasts were extracted to determine the subcellular localization of ZmMKK3 protein.The effects of 100µmol/L abscisic acid(ABA)on heat tolerance,proline content,antioxidant enzyme activity of maize seedlings,and the expression patterns of ZmMKK3 and ZmSNRK2.3 genes were analyzed under high temperature(42℃).Interacting proteins of ZmMKK3 were predicted,and the molecular mechanism of ZmMKK3 in response to high temperature stress was preliminarily explored,so as to provide genetic resources for breeding heat-tolerant maize varieties.The results showed that ZmMKK3 didn't have transmembrane domain.The secondary structure of ZmMKK3 protein primarily consisted of α-helices(36.01%),β-sheets(10.69%),and disordered regions(53.30%).ZmMKK3 was primarily localized in the nucleus and cytoplasm.Under high temperature,ABA application enhanced chlorophyll content,relative water content,proline content,and antioxidant enzyme activity[superoxide dismutase(SOD),peroxidase(POD),catalase(CAT)]in maize seedling leaves,thereby improving plant heat tolerance.ZmMKK3 gene was induced by both high temperature and ABA,its expression was stronger with ABA application under high temperature,and the expression pattern of ZmSnRK2.3 gene in the ABA signaling pathway aligned with that of ZmMKK3,indicating that ZmMKK3 induced by ABA participated in the response to high temperature stress.ZmMKK3 interacted with PP2C9(protein phosphatase 9)in the ABA signaling pathway and B4FZ26(a WD40-repeat domain protein)associated with miRNA biogenesis,and the interaction coefficients were 0.534 and 0.518,respectively.Collectively,these findings suggest that ZmMKK3 likely responds to maize high temperature stress through the ABA-MPK signaling pathway.