Isolation And Functional Analysis Of A Group A MAPK Gene ZmMPK4in Zea Mays

MAPK cascades are highly conserved modules in all eukaryotes. MAPK cascades consist of MAPK, MAPKK and MAPKKK, and transduce signals by sequential phosphorylation (MAPKKK-MAPKK-MAPK). In plants, MAPK cascades are involved in the regulation of development and growth, and programmed cell death. MAPK cascades are also in response to a diversity of environmental stimuli including cold, heat, salt, drought, UV radiation and pathogen attack. In recent years, numerous MAPKs have been isolated from different plants, and considerable progress has been made about the function of MAPKs. Though the study of MAPK signaling has been concentrated mainly in dicot plant species (Arabidopsis, tobacco, and alfafa etc.), the study on maize MAPKs is virtually lacking. Up to date, only seven MAPKs have been characterized in maize.In the present study, we isolated and characterized a MAPK gene, ZmMPK4, from maize. Sequence comparison, expression analysis and further functional studies on the overexpressing ZmMPK4transgenic tobacco were analyzed in this work. The main results are as follows:(1) We isolated ZmMPK4from maize. The ZmMPK4encodes a protein of406amino acids with a calculated molecular weight of about46.6kDa, and the ZmMPK4protein contains all11subdomains of protein kinases. In addition, the ZmMPK4consists of an adenosine5’-triphosphate (ATP)-binding region, a TEY motif and CD domain. Based on the phylogenetic tree of cloned plant MAPKs, ZmMPK4belongs to subgroup A.(2) Both the computer program prediction and transient expression of the ZmMPK4-GFP fusion protein in onion epidermal cells revealed that ZmMPK4was localized in the nucleus.(3) ZmMPK4mRNA was expressed in the leaves, stems and roots, and the transcript level in the roots was higher than that in the leaves and stems, and in the stems, its expression level was minimal. NaCl, PEG, low and high temperature stresses led to a significant increase of ZmMPK4transcript level in maize leaves. And exogenous application of ABA, SA, MeJA, ETH and H2O2also increased the ZmMPK4mRNA in maize leaves. (4) Ca2+and ROS can act as second messenger to mediate biological response. In this study, CaCb pretreatment up-regulated the ZmMPK4expression and EGTA pretreatment blocked the ZmMPK4up-regulation caused by NaCl,4℃, ABA and H2O2. And pretreatment with several ROS manipulators blocked the up-regulation caused by NaCK4℃and ABA.(5) Under low temperature and salt stresses, compared to WT plants, ZmMPK4-overexpressing lines showed a significantly higher germination rate and a better seedling growth.(6) Under low temperature stress, no obvious differences in germination rate, seedling root length and ROS accumulation were detected in WT and ZmMPK4-AF-overexpressing lines.(7) ZmMPK4-overexpressing lines displayed increased antioxidant tolerance under low temperature and salt stresses. ZmMPK4-overexpressing tobacco plants showed less ROS accumulation and higher activities of antioxidant enzymes (APX, CAT and POD) compared with WT plants under low temperature and salt stresses. In addition, under MV stress, the growth of ZmMPK4-overexpressing tobacco plants was better than the WT plants, which including more green cotyledons and longer root length.(8) Under low temperature and salt stresses, ZmMPK4-overexpressing tobacco plants accumulated more proline and soluble sugars, and increased the expression levels of ROS-related and stress-responsive genes compared with WT plants.