Isolation And Functional Analysis Of A Group C MAPKK Gene ZmMKK4 In Zea Mays

Plants are exposed to biotic and abiotic stresses throughout their life cycle. Drought and salt stress, together with low temperature, are the major factors in reducing plant growth and productivity. However, plants deploy a variety of sophisticated mechanisms to rapidly sense a changing environment and protect themselves from environmental stress. Molecular and cellular responses to these stresses have been analyzed extensively at the biochemical level: various kinds of proteins and smaller molecules, including soluble sugar, proline, and glycine betaine, accumulate. In addition, several genes known stress-responsive marker genes are induced by stresses. These observations suggest the existence of several cellular signal transduction pathways between the perception of stress signals and gene expression. Reversible protein phosphorylation, mediated by protein kinases and protein phosphatases, is the predominant regulatory mechanism in biology, modulating cellular processes such as signaling, division, growth and development.MAPK cascade is an evolutionarily conserved signaling pathway among eukaryotic organisms and plays an important role in the conversion and amplification of extracellular stimuli. The MAPK cascades are composed of MAPK, MAPKK (MAPK kinase) and MAPKKK (MAPKK kinase). They transfer signals through phosphorylation of MAPKKK-MAPKK-MAPK in turn. The number of MAPKK suggests that MAPKKs are major, multifunctional entry routes for upstream signal integration as well as bifurcation points for activation of downstream MAPKs. Several MAPKKs have been identified from different plants, however, in addition to the transcription level of ZmMEK1 was affected by salt and low temperature, much less is known about other MAPKKs fuctions in maize.In the present study, we isolated and characterized a novel MAPKK gene, ZmMKK4, from maize roots. Sequence comparison, expression analysis and further functional studies on the over-expressing ZmMKK4 transgenic plants were analyzed in this work. The main results are as follows:(1) We isolated a MAPKKgene, designated ZmMKK4, from Zea mays. The ZmMKK4 encodes a protein of 357 amino acids, and the ZmMKK4 protein contains all 11 subdomains of protein kinases. In addition, the N terminus of ZmMKK4 also contains a DEJL motif-K/R-K/R-K/R-X(1-5)-L/I-X-L/I- known to function as a MAPK docking site in plant MAPKKs. Based on the relationship tree of cloned plant MAPKKs, ZmMKK4 belongs to subgroup C.(2) The fusion protein pBI121-ZmMKK4-GFP was expressed transiently in onion epidermis. The results clearly indicate that ZmMKK4 is localized in the nucleus.(3) ZmMKK4 mRNA was expressed in the leaves, stems and roots, and the transcript level in the leaves was higher than that in the roots and stems. NaCl, 4℃and H₂O₂ treatments led to a significant increase of ZmMKK4 transcript level, and ABA declined ZmMKK4 transcript levels quickly. These results suggest that the ZmMKK4 gene is involved in response to multiple stresses and signal molecules.(4) The coding region of ZmMKK4 was introduced into the vector pBI121 under the control of the CaMV 35S promoter and then transformed into wild-type Arabidopsis (Col-0) and tobacco. The transgenic lines showed high expression of ZmMKK4 at both mRNA and protein levels by RT-PCR and Western blot.(5) Under salt stress, compared to WT plants, ZmMKK4-overexpressing lines showed a significantly higher germination rate, and seedling growth was better than WT plants.(6) ZmMKK4-overexpressing plants showed higher chlorophyll content and higher plant survival rate than WT plants under cold stress.(7) ZmMKK4-overexpressing tobacco plants showed less H₂O₂ accumulation compared with WT line under osmotic stress treatment.(8) Under environmental stress (low temperature, salt and drought), the transgenic plants accumulated more proline and soluble sugars, and showed higher activities of antioxidant enzymes compared with WT tobacco plants.(9) ZmMKK4 enhanced AtMPK4 activity of the transgenic Arabidopsis plants under cold and salt stresses. (10) Under environmental stress (low temperature, salt and drought), ZmMKK4-overexpressing plants increased the expression levels of stress-responsive marker genes.