Identification And Function Analysis In ABA-Induced Antioxidant Defense Of ZmWRKY4 Interacts With ZmMPK5 In Maize

Plants in the process of growth and development will suffer many biotic and abiotic stresses, they can restrict the normal growth of plants. Plants have many approaches against the outside damage, ABA induced antioxidant protection is an important approach. ABA can induce the growth of NADPH oxidase gene expression and the generation of reactive oxygen species (for example:H2O2), in this path ZmMPK5 play an important role. Previous studies have shown that, ZmWRKY4 play an important role in the process of plant antioxidant protection. Through yeast two hybrid and BiFC, we found that ZmWRKY4 can interact with ZmMPK5. In this experiment, we use co-immunoprecipitation (Co-IP). His-Pull down and vitro phosphorylation to identify the relationship of ZmWRKY4 with ZmMPK5. At the same time, research the influence to H2O2 and the upstream and downstream relationship in ABA signal path.We build eukaryotic expression vector ZmWRKY4-his and ZmMPK5-myc, transformed them into maize protoplasts, then conduct co-immunoprecipitation reaction. The result showed that ZmWRKY4 interact with ZmMPK5 in vivo. Purified His-ZmWRKY4 protein and had His-Pull down experiment, the result showed that ZmWRKY4 interact with ZmMPK5 in vitro. At the same time, used gel kinase reaction analysis that ZmWRKY4 is a phosphorylate substrate of ZmMPK5. By the above results, we can prove that ZmWRKY4 can interact with ZmMPK5.Treated the maize seedlings with H2O2, real-time quantitative RT-PCR analysis showed that the expression of ZmWRKY4 appear the greatest measure at 30min and 90min. Pretreatmented with reactive oxygen species scavengers substantially arrested ABA-induced gene expression. Transformed the construct vector (ZmWRKY4-YFP) into maize protoplasts, the H2O2 in protoplasts increase and ABA treatment can enhance this phenomenon. ZmWRKY4 was silenced using the RNA interfere technology, the H2O2 in protoplasts decrease and ABA treatment can’t reverse this phenomenon. Transformed the construct vector (ZmWRKY4-YFP) into maize protoplasts, real-time quantitative RT-PCR analysis showed that the expression of NADPH oxidase gene increase, on the contrary, silence the ZmWRKY4 resulted in the decrease of NADPH oxidase geneexpression. From this we can see that ZmWRKY4 take part in the ABA induced antioxidant protection and in this way H2O2 is required.Used protoplast transient expression system, overexpression ZmMPK5 and silence ZmWRKY4 can induce H2O2 accumulation、activities and gene expression of antioxidant enzymes SOD and APX decrease. On the contrary, overexpression ZmWRKY4 and silence ZmMPK5 can induce H2O2 accumulation、activities and gene expression of antioxidant enzymes SOD and APX increase. These results show that ZmMPK5 is on the upstream of ZmWRKY4 in the ABA induced antioxidant protection.