Maize(Zea Mays.L) ZmMKK10 Positively Regulates The Process Of Cell Death

Plants have evolved sophisticated signals transduction network and response to complex and changeable environments. Mitogen-activated protein kinase (MAPK) cascades, which are highly conserved signal transduction systems,play vital roles in regulating plant growth,development and stress responses. Typical MAPK cascades contain MAPKKK-MAPKK-MAPK and transduce signals by sequentially phosphorylating and activating. Compared with Arabidopsis or rice, there is less reports on maize MAPK cascades. In this study, we reported the physiological function of ZmMKK10, a group D MAP kinase kinase in maize, in regulating positively the process of cell death by using maize mesophyll protoplast system and the model plant Arabidopsis.Using the conserved sequences of 10 Arabidopsis MKKs as queries to maize B73 genome, we found a group D MKK, named as ZmMKK10. Analysis of transcript level of ZmMKK10 in different tissues by Q-PCR revealed that the transcripts of ZmMKK10 could be detected in all tested tissues.Transient expression of ZmMKK10 in maize mesophyll protoplast suggested that ZmMKK10 localized predominantly in cytoplasm. Kinase activity analysis of recombinant ZmMKK10 showed that the Mg2+ion was required for its kinase activity.Using constitutively active or inactive forms of MKKs to explore their function has been a powerful strategy in MAPK cascade studies. ZmMKK10DD (mimic constitutively active form of ZmMKK10) and ZmMKK10KR (mimic constitutively inactive form of ZmMKK10) were obtained by site-directed mutant PCR to explore the physiology function of ZmMKK10. Establishment of maize transgenic plants overexpressing ZmMKK10WT (wild type ZmMKK10) or ZmMKK10DD was failed. In this study, we found that transient expression of ZmMKK10WT or ZmMKK10DD in maize mesophyll protoplast could accelerate remarkably the cell death rate of protoplast, while ZmMKK10KR could not.These results suggested that activated ZmMKK10 may positively regulate the process of cell death in maize.Since no transgenic maize plants, model plants Arabidopsis was used to analysis the regulatory mechanism of ZmMKK10 in cell death and then constructed ZmMKK10WT, ZmMKK10DD and ZmMKK10KR transgenic Arabidopsis plants under inducible promoter. After DEX treatment,ZmMKK10WT and ZmMKK10DD plants showed wilt, ion leakage and other cell death phenomena, while ZmMKK10KR and Vector plants did not exhibit any of the above phenotypes. Further study of ZmMKK10DD/mpk6 and ZmMKK10DD/mpk3 plants confirmed that ZmMKK10 induced cell death through activating MPK6/MPK3 and was accompanied with ethylene production and H2O2 accumulation. Pharmacological analysis with ethylene synthesis inhibitors and H2O2 scavenger showed that the process of cell death induced by ZmMKK10 was partially dependent on ethylene synthesis and H2O2 accumulation. These data suggested that ZmMKK10-MPK6/MPK3 induced cell death mediated by ethylene synthesis and H2O2 accumulation.In order to find the downstream MPKs of ZmMKK10 in maize, we used yeast two hybrid to screen which one interacted with ZmMKK10 among 20 ZmMPKs. Further in vitro phosphorylation analysis showed that ZmMPK3 and ZmMPK7, which were respectively orthologues of Arabidopsis MPK3 and MPK6, were directly phosphorylated by ZmMKK10 in vitro. These data suggested that ZmMPK3 and ZmMPK7 may be the downstream MPKs of ZmMKK10 in the process of cell death in maize.In conclusion, we found that ZmMKK10, a group D MKK, positively regulate the process of cell death, and ZmMPK3/ZmMPK7 may be the downstream MPKs in maize. This finding supplements the study of MAPK cascades in maize and provides a basis for further study of the function of ZmMKK10.