Isolation And Functional Identification Of A Novel Protein Phosphatase Gene, ZmPP2C2, In Maize Roots And Responses Of Transgenic Tobacco To Environmental Stress

Abiotic stresses such as low temperature, drought and high salt, influence plant growth, productivity and development. To cope with unpredictable environmental changes, plants evolve signal transduction mechanisms by which they receive stress signals and regulate their stress-tolerant reactions. Molecular and cellular responses to these stresses have been analyzed extensively at the biochemical level: various kinds of proteins and smaller molecules, including sugars, proline, and glycine betaine, accumulate, in addition, many 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. Yet the mechanisms underlying the perception and transduction of stress signals manifested in development of stress tolerance are just being revealed.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. Protein phosphatases, by opposing the action of protein kinases, provide modulation and reversibility of the phosphoregulatory mechanism. Biochemical and molecular genetic studies have identified PP2Cs as regulators of stress. However, knowledge of the transduction pathways, especially in stress responses, is still incomplete. The involvement of PP2Cs in stresses acclimation is also far from clear.In this study, we isolated a PP2C gene (ZmPP2C2) from Zea mays roots and characterized its expression patterns in different tissues and in response to abiotic stresses and signal substances. We show that ZmPP2C2 was basically expressed in the roots, stems, leaves and seeds, especially in seeds. Moreover, we found that ZmPP2C2 exhibited different responses to diverse abiotic stresses and signal substances, and revealed a quick and transient response to low temperature. Then, to study the contribution of PP2Cs to stress signaling we generated 35S::ZmPP2C2 transgenic tobacco. The transgenic plants showed enhanced tolerance to cold, drought and salt than wild type tobacco. The main results are as follows:1. In Southern blot analysis, hybridization of the ZmPP2C2 probe with genomic DNA of Zea mays reveals that the ZmPP2C2 gene is present in the Zea mays genome as a single copy. Northern blot analysis revealed that this clone was expressed in roots, stems , leaves and seeds, and the transcript were relatively more abundant in seeds than in other organs. Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from Zea mays according to the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The 5'and 3'fragment of the cDNA was isolated by 5'and 3'RACE. The clone, which named ZmPP2C2 (Acession Numeber:AY830123), contains 1399 bp nucleotides with an open reading frame (ORF) of 855 bp comprising 284 amino acid residues with the predicted molecular mass of 30.9 kDa. The deduced amino acid sequence showed high identities with PP2C from Arabidopsis, Oryza sativa, Medicago and Zea mays. Amino acid sequence alignment revealed that six of the seven residues that are putatively involved in the coordination of the phosphate and metal ions were present in the sequence, and the eleven conserved motifs presented in all Ser/Thr PP2Cs were found in the catalytic domain. Software analysis showed that ZmPP2C2 protein was a hydrophilic protein, which contained one typical superhelix from outside to inside and two potential superhelixes from inside to outside.2. ABA and low temperature significantly increased the transcript levels of ZmPP2C, and Ca2+ acts as a negative regulator in ABA-induced ZmPP2C2 transcription. Besides, ZmPP2C2 response to low temperature at both transcription and translation level.3. The pBI121- ZmPP2C2-GFP fusion protein is clearly targeted to the nucleus in a transient transfection assay. Construction of a dendrogram based on the homologous full-length amino acid sequences revealed the close imilarity between ZmPP2C2 and the PP2C from Arabidopsis and Medicago.4. A recombinant of prokaryotic expression vector pET- ZmPP2C2 was constructed and expressed in E.Coli. BL21. The strong induced fusion protein were purified and used to immunize white mice to obtain antiserum. Western hybridization revealed the presence of the strong positive protein signals corresponding to ZmPP2C2 in transgenic tobacco.5. The full-length ZmPP2C2 cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense constructs. The constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method and verified by PCR , Northern blot and Western blot. It was indicated that the ZmPP2C2 gene had been recombined into tobacco genome and transgenic tobacco plants were obtained.6. Expression of the ZmPP2C2 gene attenuated the delay in germination at low temperature, salt, and drought stress, and improved the germination rate in transgenic tobacco.7. Under salt and drought stress, Pn in both wild type tobacco and transgenic tobacco decreased, but Pn decreased less in transgenic tobacco compared with wild type tobacco. There was no obvious difference between wild-type and transgenic plants about the change of Ci and Gs. However, the level of Fv/Fm in transgenic tobacco decreased less compared with wild type tobacco under salt stress. It indicated that PSâ…¡of transgenic tobacco plants suffered less injury compared with wild type tobacco.8. Under environmental stress (low temperature, salt and drought), the relative electrical conductivity and MDA content in both wild type and transgenic tobacco plants increased, which resulted in the increase of membrane permeability and electrolyte leakage, and the structure and function of cell membrane was damaged . However the degree of increase in transgenic tobacco plants was lower than that in wild-type tobacco plants, which indicated that the extent of the membrance lipid perioxidation in transgenic tobacco was lighter than that in wild type tobacco. It is favorable to maintain the function of cell membrane.9. Under cold stress , the activities of SOD and CAT increased in both wild types and transgenic tobacco plants, while the activitie of POD increased first and then decreased in both wild types and transgenic tobacco plants. However ,Under salt stress , the activitie of SOD increased first and then decreased in both wild types and transgenic tobacco plants, and the activities of POD and CAT increased in both wild types and transgenic tobacco plants. Interestedly, the activities of SOD , POD and CAT increased first and then decreased in both wild types and transgenic tobacco plants. Furthermore, in all stresses the transgenic tobacco plants always sustained higher activities of both SOD, POD and CAT.10. Under environmental stress (low temperature, salt and drought), the transgenic tobacco plants accumulated more proline , soluble sugars and soluble protein compared with wild type tobacco plants.