Genome-wide Characterization Of Mitogen-activated Protein Kinase Gene Family And Functional Analysis Of ZmMPK4 Gene Pair

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes. A MAPK cascade consists of MAPKKK-MAPKK-MAPK, that sequentially phosphorylate the corresponding downstream substrates. Each gene family of MAPKKK, MAPKK or MAPK is conserved in different plant species. The maize genome has been sequenced to date. It is possible to dissect the information of maize MAPK gene family based on the genomic sequence. MAPK links upstream components to downstream substrates. Previously, it has been reported that MAPK cascades participated in ABA signaling. However, the specific MAPK gene in ABA signaling is still sparse.In this study, we identified MAPK genes in the maize genome based on the conservation of plant MAPKs. The classification, the protein properties, the gene structure, the chromosomal distribution, the evolutionary relationship and the expression pattern in maize roots, stems and leaves of maize MAPK genes have been analyzed. Based on the analysis, we focused on the function of ZmMPK4. ZmMPK4 is a two-copy gene and exists as gene pair with ZmMPK3. ZmMPK4 plays a role in ABA signaling. The main results are as follows:(1) Using database BLAST and Stand-alone BLAST, we identified 19 MAPK genes in the maize genome. The amino acids of 19 MAPK proteins are between 369 and 642. The molecular weight of 19 MAPK proteins are between 42.199 kDa and 72.444 kDa. The pI of 19 MAPK proteins are between 5.26 and 9.82. Four of the 19 MAPK genes exist in chromosome 6 or 8. Three exist in chromosome 5 or 9. Two exist in chromosome 10. One exists in chromosome 1, 3 or 4. The maize MAPKs share more evolutionary relationship with rice MAPKs than Arabidopsis or poplar MAPKs.(2) ZmMPK1 shares a high sequence similarity with ZmMPK2. There is no EST sequence specifically matches ZmMPK1 sequence. RT-PCR analysis shows that there is not detectable mRNA in roots, stems or leaves of maize seedlings, suggesting ZmMPK1 might be a pseudogene. Other 18 genes can be detected in roots, stems or leaves of maize seedlings. In addition to ZmMPK12, ZmMPK18 and ZmMPK19, other 15 genes expressed in different level.(3) PCR analysis shows that the band of ZmMPK4 did not match the band of previously reported ZmMPK4 (ZmMPK4-2 in our study). ZmMPK4-2 is a alternative transcript of ZmMPK4 gene that can take alternative splicing. Gene sequence and MAPK gene family analysis revealed that ZmMPK4 is a two-copy gene and exists as gene pair with ZmMPK3. Southern blot showed ZmMPK4 is a two-copy gene. The nucleotide sequences of ZmMPK3 and ZmMPK4 share similarity of 92.1%. The amino acid sequences of ZmMPK3 and ZmMPK4 share similarity of 90.0%. ZmMPK3 and ZmMPK4 are located on the short arm of chromosome 1 and the long arm of chromosome 9, respectively. Both ZmMPK3 and ZmMPK4 could generate mRNA and proteins.(4) The third intron of ZmMPK4 gene is GC-AG type. ZmMPK4-2 is one alternative transcript result from retention of the third intron of ZmMPK4 gene. The transcript of ZmMPK4-2 is low and mainly found in maize leaves. Another alternative transcript, the main transcript, of ZmMPK4 gene is ZmMPK4-1 without the third intron. The third intron of ZmMPK3 gene is GT-AG type.(5) Northern blot analysis indicated that ZmMPK3 is mainly expressed in leaves of 5-d-old seedlings and ZmMPK4 in roots. ABA (100 ?M) or NaCl (200 mM) up-regulated the expression of both ZmMPK3 and ZmMPK4 with different patterns. Northern blot analysis did not differentiate the expression of ZmMPK4-1 and ZmMPK4-2. RT-PCR using specific primers revealed that the alternative splicing predominantly occurred in leaves of 5-d-old seedlings. The alternative splicing is regulated by ABA or NaCl.(6) ZmMPK3 antibody (Anti-ZmMPK3) was generated. Generation of specific antibodies for ZmMPK3 and ZmMPK4 is impossible because of the high similarity of amino acid. In leaves of 5-d-old seedlings, Anti-ZmMPK3 could detect protein band with an approximate molecular mass of 43 kDa. The protein amount could slightly be induced by ABA (100 ?M). Immunoprecipitation assay showed that ABA (100 ?M) stimulates a rapid activation of the 43 kDa protein in 0.5 h or 1 h. Transient expression in Nicotiana benthamiana was performed to specifically examine the kinase activities of ZmMPK3, ZmMPK4-1 or ZmMPK4-2. The results showed that ABA (100 ?M) activated ZmMPK3 or ZmMPK4-1, but not ZmMPK4-1 in 0.5 h, 1 h or 2 h.(7) Transiently expressing of 35S-GFP-ZmMPK fusion proteins suggest that ZmMPK3 or ZmMPK4-1 fusion protein localized in cell membrane and nucleus, and ZmMPK4-2 fusion protein localized in cell membrane and cytoplasm. These results indicate the insertion of 30 amino acids may disturb the localization of ZmMPK4 protein.(8) Overexpressing of ZmMPK4-1 in Arabidopsis accelerated stem development. In the adult plants, the rosette leaves arose from the stem and can form stem subsequently. The wild-type and ZmMPK4-1-overexpressing Arabidopsis showed no difference in germination (about 100%) on MS media without ABA treatment. ABA (0.5 ?M) inhibited the germination of both wild-type and ZmMPK4-1-overexpressing seeds. The inhibition effect of ABA on ZmMPK4-1-overexpressing seeds was stronger than that of ABA on wild-type seeds. Immunoprecipitation assay showed anti-p-ERK could detect kinase activity in ZmMPK4-1-overexpressing plants under normal growth conditions. ABA (0.5 ?M) stimulates activation of ZmMPK4-1 in 30 min. AtACT1 is upregulated in ZmMPK4-1-overexpressing plants under light growth conditions. Furthermore, in ZmMPK4-1-overexpressing plants, ABA-inducted AtACT2, ABI3 or ABI5 expression was enhanced.