| Programmed cell death,also known as apoptosis, means:a death way that forwardly, orderly, and genetic control of cell, in order to growth and development better and adapt to the environment by growing or response the suffering from biotic or abiotic stress around.Programmed cell death involves many aspects of plant growth and development, such as senescence, the margin formation of leaf, duct formation, reproductive-related unisexual flower formation or something else related with reproduction, pollen sterility, endosperm disappear in the seed germination, the outermost layer of dead cells which in order to protect root cap cells, are all closely associated with apoptosis.Recent studies have also found that stress could induces apoptosis, when tomato leaf infected by bacteria, PCD will take place along the bacteria plaque, the formation of necrotic lesions can prevent the leaf to further infection of bacteria, causing light in Arabidopsis protoplasts occurred apoptosis, water stress induced apoptosis occurs in Arabidopsis root tip, lead and other heavy metal ions cause root tip occurred PCD. In order to survive the plant, a increased or decreased apoptosis can help plant to adapt to the ambient better.ZmCCaMK is a Ca2+ /CaM -dependent protein kinase, has an important role in many aspects of plant growth and development. It has been shown ZmCCaMK is an important signaling pathway in plants, and have crosstalk with the presence of MAPK signal and ZmCCaMK may be involved in antioxidant defense in plants. CAMK?, the homologs gene in animals, have been found that meybe involved in the process of apoptosis, overexpression CAMK?, can significantly reduce the apoptosis of nerve cells.So whether ZmCCaMK play a role in a plant cell apoptosis or not?We first deal maize leaf protoplasts by PEG6000 simulated drought stress, stain with FITC-PI and test the apoptosis by flow cytometry, then apoptosis rate rise over time was found, TUNEL staining and confocal showed positive staining, extract genomic DNA of protoplast to test DN A ladder by agarose gel electrophoresis and detected a significant DNAladder, from methods above, we found PEG6000 simulated drought stress can induce the occurrence of maize protoplasts programmed cell death.Next, we use maize protoplasts homeopathic expression system, overexpression and silencing ZmCCaMK, by FITC-PI staining and flow cytometry we discovered the apoptosis rate of ZmCCaMK over-expression group was significantly lower than the control group in the caseof drought stress; but the silent treatment group was significantly higher than the control treatment group, which we judge ZmCCaMK might be involved in drought stress-induced PCD occurs in maize protoplasts. To further validate these conclusions, we use OsDMI3 rice mutants which highly homologous with ZmCCaMK do the the following experiments,.we found that the PCD fraction of mutant protoplasts tested by flow cytometry showed consistent with the previous conclusion, the apoptosis of mutants treated group increased significantly; take stems of wild-type and mutant rice that treated Oh,12h, 24h,48h,96h, frozen sections, and use TUNEL immunofluorescence in situ, observed the case of staining and found that mutant fluorescent staining treatment group were higher than the control treatment group. Further validate the above conclusions.Studies have shown antioxidant defense enzymes APX environment and the formation of apoptosis occurring enzyme activity decreased, easier to form the environment of apoptosis, NO and apoptosis-related enzymes nitration ubiquitination related. We further determined maize protoplasts H2O2, and NO content and antioxidant defense enzymes APX, SOD, CAT enzyme activity. Experiments show that after treatment, overexpression ZmCCaMK can significantly increase the antioxidant activity of protective enzymes, the silencing activity decreased significantly, indicating that indeed there is a relationship between the two, is also associated with apoptosis need further validation. |
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