Molecular Cloning, Expression, Localization And Function Analysis Of ABA-Induced MAPK From Leaves Of Maize (Zea Mays L.) Plants

The phytohormone abscisic acid (ABA) plays regulatory roles in plant growth and development processes. ABA accumulation and subsequent physiological effects stimulation require a complex interplay of signaling cascades. The mitogen-activated protein kinase (MAPK) cascades have been reported to function in various signal transduction pathways in eukaryotes. It is very important to study MAPK function in abscisic acid signal transduction.Our lab previous studies reported that a 46 kDa MAPK (p46MAPK) was involved in ABA-induced antioxidant defense in leaves of maize (Zea mays) plants. However, the identity of the p46MAPK in maize is not clear. It is important to reveal the identity and characterization for further research at molecular level.According to the homologous sequences from other plant MAPK, the degenerate primers CGCYTAYGGVATCGTYTGCTC and GTDACVACRTAYTCMGTCAT which correspond to the conserved MAPK domains:-GAYGIVCS- and -MTEYVVTRW-, respectively, were used to amplify by PCR the cDNA encoding MAPK-like from maize leaves treated with ABA. Two degenerate primers AARATHGCNAANGCNTTYGA and GTDACVACRTAYTCMGTCAT which correspond to purifed fragments of ABA-induced p46MAPK:-KIANAFD- and -MTEYVVTRW-, respectively, were used to amplify by PCR the p46MAPK cDNA. Then, a combination of an in silico comparative cloning with a PCR-based strategy was developed to obtain new MAPK genes. We identified two MAPK genes, designated ZmMPK3 and p46MAPK (ZmMPK5). Sequence analysis showed that the two MAPKs consisted of an ATP-binding region, a MAPK family signature, a catalytic loop, a phosphorylation motif (TEY motif) in activation loop, and CD domain. The protein exhibits closest homology to an ERK subgroup of plant MAPKs.In order to study the functions of ZmMPK3 and ZmMPK5, the ORF of the two MAPKs were inserted into the pGEX-4T vector as GST fusion proteins. The constructs were transformed into E.coli BL21. Expressions were induced by the addition of IPTG. The 71 kD GST-MAPKs fusion proteins were expressed and purified by affinity chromatography. The two fusion proteins expressed in vitro have no kinase activation by the measure of solution activation. The polyclonal antibodys against the C-terminal region of ZmMPK3 and ZmMPK5 were made. The results show that ZmMPK5C antibody and ZmMPK3c antibody have good specificity.Immunoprecipitation and in-gel kinase assay further confirm that ABA- and H2O2-induced 46 kD MAPK is ZmMPK5.100μM SA,1 mM ETH,10% PEG,250 mM NaCl,500μM CdCl2, low temperature, wounding and UV are able to mediate the transcript levels and kinase activities of ZmMPK3 and ZmMPK5.To investigate subcellular locations of the two MAPKs, ORFs of the MAPKs were inserted into sites of pXZP008 vector. The constructs (35S-ZmMPK3/ZmMPK5-YFP) were transformed into maize protoplasts. The results showed that ZmMPK3/ZmMPK5 was mainly localized to the cell nucleus. Using protoplast transient expression assay, over-expression of ZmMPK3 increased the transcription level of cAPX, SOD4 and up-regulated enzyme activities of SOD and cAPX. We confirmed that ZmMPK3 is involved in ABA-induced antioxidant defense.