Isolation And Functional Analysis Of A Group C MAPK Gene ZmMPK7 In Zea Mays

The 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. In plant, MAPKs are classified into four major groups designated A-D according to their sequence homology. Many MAPKs belonging to groups A and B have been studied. Little information, however, is available for group C MAPKs, although recent studies have demonstrated that some group C members are in response to environmental stress and hormonal stimuli, including ABA and H2O2. Since the responses of some group C MAPKs in different species have been characterized, there is evidence that they may have similar functions across species. However, no data regarding group C MAPKs from maize are currently available. There are also no available data that detail how they mediate ABA or H2O2 signaling. In particular, the relationship between group C MAPKs and antioxidant defense systems warrants further research. In the present study, a series of studies based on the materials of Ludan50 seedlings (Zea mays L.) have been conducted on the ZmMPK7 isolation, sequence comparison, expression analysis and further functional studies on the over-expressing ZmMPK7 transgenic tobacco. The main results are as follows:1. Collecting and analyzing MAPK genes information of Zea mays from GenBankAccording to the sequences of the 20 Arabidopsis MAPK genes, 39 pieces of information on gene accession have been identified from GenBank. Among them, some are repeated in completed sequences or in partial sequences. After the analysis, 12 specific gene completed sequences were reserved and can be classified into four groups, A-D. Most of the members belong to the Group C and D, which suggests that the diversity of these MAPKs may related to the specific activation of signal transduction.2. Isolation and characterization of a group C MAPK gene ZmMPK7 from Zea mays According to the sequences analysis about the MAPK genes information in maize, the specific primers were designed and a DNA fragment was amplified from the maize seedlings. The isolated fragment of the MAPK gene from maize contains a 1113 bp ORF, which encodes a protein of 370 amino acids with a calculated molecular weight of about 42.5 kDa. An alignment of the amino acid sequences of ZmMPK7 and MAPKs from other plants shows that the ZmMPK7 protein contains all 11 subdomains of protein kinases with serine/threonine specificity and the TEY motif. A phylogenetic tree analysis indicates that it is most homologous to Oryza sativa OsMSRMK3, Arabidopsis thaliana ATMPK1, ATMPK2, ATMPK7 and ATMPK14, Nicotiana tabacum NtNTF3, and Pisum sativum PsMPK2, all of which are classified as group C MAPKs.3. Expression analysis of ZmMPK7 in Zea maysIn order to identify the stressors that elicit a response from ZmMPK7, Northern blot analysis was carried out firstly. The ZmMPK7 gene had a relatively high level of basal expression. An increase in the expression of ZmMPK7 was detected in samples treated with 100μM ABA or 1.5 mM H₂O₂, but the samples exposed to 15% PEG-6000 or 4oC demonstrated no obvious increase in ZmMPK7 expression. ABA and H₂O₂-induced expression of ZmMPK7 was time-dependent in a Ca²⁺-dependent way. Semi-quantitative RT-PCR analysis indicated that several ROS manipulators, including imidazole, an inhibitor of NADPH oxidase, and the H2O2 scavengers Tiron and dimethylthiourea (DMTU) may inhibite the upregulation caused by ABA to varying extents. The basal ZmMPK7 mRNA level was relatively stable across the photoperiods and no rhythmic expression is detected in maize seedlings.4. Over-expressing ZmMPK7 in Nicotiana tabacum provides protection against ROS-mediated injury under osmotic stressTo further test the roles of ZmMPK7 in ABA and H2O2-mediated signaling, ZmMPK7 was over-expressed in transgenic tobacco plants under control of the CaMV 35S promoter. Homozygous T3 seeds of transgenic tobacco and wild-type lines were selected for the physiological analysis. It revealed that ZmMPK7-overexpressing tobacco plants showed less H2O2 accumulation and less MDA content compared with wild-type lines under osmotic stress treatment. After PEG-6000 treatment, no obvious differences in total SOD and CAT activities were detected between wild-type and transgenic tobacco plants. However, differences were detected in total peroxidase (POD) and ascorbic acid peroxidase (APX) activities. In the ZmMPK7-overexpressing tobacco lines, total ascorbic acid and dehydroascorbic acid contents were detected higher than those in wild-type tobacco. It is reasonable to speculate that the enhanced antioxidant system in transgenic tobacco plants may contribute to the protection against ROS-mediated injury under osmotic stress. Moreover, Methyl viologen (MV) in different concentration was used to estimate the effects of the oxidative stress on the growth of the wild-type and transgenic tobacco plants. It showed that the transgenic tobacco lines grew better under both high and low MV concentrations. This suggests again the advantages of anti-oxidative characterization in the ZmMPK7-overexpressing tobacco lines.5. ZmMPK7 is localized in the nucleusBioinformatics analysis showed it occupied the nucleus localization characterization. Transient expression of ZmMPK7-GFP fusion gene in onion (Allium cepa) epidermal cells confirmed that ZmMPK7 is a nuclear-localized protein.6. Isolation and characterization of a MAPKK gene ZmMKK3 in Zea maysSimilar analysis about the putative sequence information of maize MAPKK in GenBank was conducted. We isolated a specific DNA fragment from maize seedlings, which was speculated to encode a MAPKK. The isolated fragment contains a 1572 bp ORF, which encodes a protein of 523 amino acids and it was designated as ZmMKK3. An alignment of the amino acid sequences of ZmMKK3 and MAPKKs from other plants shows that the ZmMKK3 protein contains all the subdomains of the plant MAPKKs and the consensus sequence S/TxxxxxS/T. A phylogenetic tree analysis indicates that it is most homologous to Oryza sativa OsMKK3 and Arabidopsis thaliana AtMKK3, all of which are classified as group B MAPKKs. It has been indicated in a previous report that the group B MAPKK member, AtMKK3, is upstream of AtMPK7. So we speculated that ZmMKK3 may also interact with ZmMPK7. Further confirmation is required.