The Study On The Regulatory Mechanism Of Plant Cell Death By Extracellular ATP Under Stress

Adenosine triphosphate(ATP) not only in form of the energy currency of existence within the cells, and also exists in animal and plant extracellular matrix widely as signaling molecules. Extracellular ATP(eATP) bind to the receptors on the surface of the cell membrane, and stimulate the intracellular second messenger(Ca2+,H2O2), which regulates a variety of physiological activities of plant cells. By using the tobacco(Nicotiana tabacum L. cv. Bright Yellow-2) suspension cells as the experimental materials, the role of extracellular ATP in the SA, Cu2+, NaCl-induced cell death and the possible mechanism were studied. Mainly have the following findings:(1) Extracellular ATP alleviate the cell death induced by plant hormone salicylic acid(SA). High concentration of SA induce cell death, the addition of exogenous ATP can alleviate SA-induced cell death. At the same time, it was found that SA could reduce the activity of NADPH oxidase, while exogenous ATP could increase. In order to further explore the regulatory mechanism of eATP, and verify that the pathway is related to NADPH oxidase or not, we treated cells with the inhibitor of NADPH oxidase — diphenylene iodide(DPI). The results showed that DPI could inhibit the alleviative effect of eATP on the SA-induced cell death, so the regulation of the signal pathway may be related to the NADPH oxidase.(2) Extracellular ATP affect the cell death and the production of H2O2 induced by copper. High concentration of Cu2+ induce cell death and increase the intracellular and extracellular H2O2 levels. With the addition of DMTU — scavenger of H2O2, Cu2+induced cell death returned to the control level, which indicates that the production of H2O2 is the most important reason of Cu2+ caused cell death. Cu2+ also increased the activity of NADPH oxidase. By adding DPI inhibited NADPH oxidase, the Cu2+induced cell death and intracellular and extracellular H2O2 production were alleviated,but did not return to control levels. It's indicating that Cu2+ induced cell death and H2O2 production are related to NADPH oxidase. Addition of exogenous ATP increased the cell death, the production of intracellular and extracellular H2O2, and theactivity of NADPH oxidase. But were abolished with the pretreatment of DPI. In conclusion, Cu2+ induced cell death mainly relate to the content of intracellular and extracellular H2O2, and the activity of NADPH oxidase. This indicates that eATP regulates the Cu2+-induced cell death and intracellular and extracellular H2O2 generation by stimulation of NADPH oxidase.(3) Extracellular ATP affect cell death and respiration induced by NaCl. High concentration of NaCl induced cell death and respiratory inhibition, and reduce the level of eATP content. The study also showed that decline of the eATP level could lead to cell death. In order to explore the relationship of eATP and NaCl-induced cell death, added exogenous ATP in NaCl treated cells. The results show that cell death,respiratory depression, eATP content decline were alleviated with the addition of exogenous ATP. This indicates that eATP regulates NaCl-induced cell death and respiratory decrease.In conclusion, as an important signal molecule, eATP regulates plant cell activity,by affecting NADPH oxidase and H2O2. It has important physiological significance to maintain the normal physiological activities of plants and the regulation of cell resistance under environmental changes.