Identification Of The Interaction Between ZmMAPK5 And Its Target Proteins And Functional Analysis Of ZmPPa4 Transgenic Tobacco Under Salt Stress

MAPK(Mitogen-Activated Protein Kinase)is one of the important members in MAPK cascade signaling pathway,and functions as kinase or substrates.Using ZmMAPK5 as a bait to screen the cDNA library of maize leaves in previous study,and some positive clones were obtained.The positive clones were sequenced,and homology alignment analysis was performed by BLAST.There were 5 putative proteins interacted with ZmMAPK5:ZmPPa4(Soluble inorganic pyrophosphatase),ZmNAC84(NAC transcription factor),ZmRNP(Ribonucleoprotein),ZmTPI(Triosephosphate isomerase)and ZmALT(Alanine aminotransferase2).MAPK plays a crucial role in responding to both biotic and abiotic stresses,in order to further study the functions of MAPK,researches are as follows:First,the five target genes were subcloned into the target vector,and the interaction between ZmMAPK5 and its target proteins was verified using in vivo and in vitro assays:yeast two-hybrid assay,GST pull-down,and Co-immunoprecipitation(Co-IP).The results showed that ZmMAPK5 interacted with ZmPPa4,ZmNAC84 and ZmRNP both in vivo and in vitro,while ZmMAPK5 did not interact with ZmTPI or ZmALT.ZmPPa4 belongs to a soluble inorganic pyrophosphatase,which acts as a proton pump to maintain normal physiological and biochemical reactions in the cytoplasm.Previous studies showed that soluble inorganic pyrophosphatase could involve in the response to biotic and abiotic stresses in plants,such as cold,salt and drought stress.To analyse the function of ZmPPa4 preliminary,ZmPPa4-YFP was transiently overexpressed in tobacco leaves by agrobacterium-mediated transformation and its subcellular localization was observed by laser confocal microscopy.The results showed that ZmPPa4 was localized in the cell membrane and cytoplasm.Secondly,in order to verify whether ZmPPa4 can be phosphorylated by ZmMAPK5,prokaryotic expression and purification of GST-ZmMAPK5 and His-ZmPPa4 protein were used to perform the in vitro kinase assay,results showed that ZmPPa4 can not be phosphoiylated by ZmMAPK5 in vitro,and ZmPPa4 might not be a phosphorylation substrate of ZmMAPK5.Using transient expression system of maize protoplasts to determine whether ZmPPa4 functions in antioxidant defense,the results showed that transient overexpression of ZmPPa4 significantly increased the activities of antioxidant defense enzymes APX and SOD,ZmPPa4 was involved in antioxidant defense.Finally,to further investigate whether ZmPPa4 is involved in salt stress,we constructed ZmPPa4 overexpressed tobacco by agrobacterium-mediated genetic transformation,observed its growth phenotype and tested its physiological and biochemical indicators under salt stress.After treatment with 400 mM NaCl for 10 days,compared with wild type,ZmPPa4 overexpressing plants showed the increased tolerance to salt stress,the improved antioxidant defense enzyme activities,the raised relative water contents,the decreased MDA contents and the rate of electrolyte leakage.The above results indicated that ZmPPa4 was involved in the response of plants to salt stress and enhanced the tolerance of plants to salt stress.