Role Of Extracellular ATP In Salicylic Acid-regulated Plant Cell Death And Respiration

ATP(Adenosine 5’-triphosphate, ATP) not only exists in the interior of the cell but also presents in the extracellular matrix of animal and plant cells. Extracellular ATP(extracellular ATP) can be used as a signaling molecule to stimulates specific intracellular second messengers(such as Ca2+, H2O2, NO), which regulates many physiological responses of cells. Salicylic acid(Salicylic acid, SA) is one of the most important signaling molecules in plant. the regulation role of SA has been reflected mainly in the following two aspects: 1) the rise SA at certain level can induce plant resistance to viruses and pathogens as an important signal; 2) higher levels of salicylic acid can induce programmed cell death. And many forms of cell death, such as those induced by some biotic and abiotic factors(such as pathogen infection, ozone treatment and salt stress, etc.), could is related to SA.here, We use Tobacco((Nicotiana tabacum L.BY-2) suspension cells as experimental materials to study Role of extracellular ATP in Salicylic acid-regulated plant cell death and respiration. The major results were shown as below:(1) Treatment of tobacco suspension cells with different concentrations SA(0 μmol, 100 μmol, 300 μmol,500 μmol, 700 μmol) obviously caused BY tobacco-2 cell death. The levels of cell death increased with the increase in the concentration of exogenous SA and increased with the increase of treatment time. FDA staining was used as a qualitative tool to visualize the SA-induce cell death. Microscopic observation showed that FDA fluorescence decreased with the increase in exogenous SA concentrations.(2) Treatment with exogenous 500 μmol SA for 10 h significantly decreased the levels of iATP and this exogenous SA also decreased the eATP levels. It was found that when this exogenous SA leaded to cell death, this exogenous SA also led to a serious inhibition in cell respiration and the decreases in the levels of both iATP and eATP levels. These results indicate that when the respiratory substrate unchanged, treatment of the cell cultures with exogenous SA decreased respiratory O 2 uptake in a dose-dependent manner, which is similar to the change in the levels of eATP and iATP after the treatment with exogenous SA.(3) The tobacco suspension cells were treated with exogenous AMP-PCP(β,γ-methyleneadenosine5’-triphosphate), a non-hydrolysable analogue of ATP. These results suggest that the replacement of eATP with AMP-PCP can initiate a cell death in tobacco cell cultures. To examine whether the induction of cell death by SA is linked to the depletion of eATP, we added exogenous ATP to the SA-treated tobacco cell cultures to limit the reduction of eATP due to the application of SA. The results showed that the added exogenous ATP can alleviatethe SA-induced cell death, indicating that the SA-induced cell death could be associated with a decrease in eATP level and eATP plays a vital role in regulating the SA-induced cell death.(4) To examine the internal mechanism for the role of eATP in regulating the SA-induced cell death Ca2+ inhibitors EGTA(Ethylene glycol tetraacetic acid, Ca2+ chelating agent), Gd Cl3(Gadolinium chloride, Ca2+channel inhibitors) and exogenous Ca2+ were used. The result shows that the application of GdCl3 or EGTA themselves had no significant effects on any of the experimental parameter. But, the addition of GdCl3 or EGTA almost abolished the alleviative effect of eATP on the SA-induced decreases in cell viability and respiration. This result suggests that the effects of eATP on the SA-induced cell death and the inhibition of respiration could also be partly attributed to a positive regulation of the mitochondrial functions by Ca2+.The research results showed that cell death and respiratory decrease induced by SA has certain internal relation with the eATP decrease. SA could decrease the mitochondrial ATP production by impairing mitochondrial respiratory metabolism and subsequently lead to the decline of e ATP levels. Decline in the intracellular ATP production could cause the decrease in the extracellular ATP. The SA-induced cell death is a result of a decline of iATP production due to the inhibition of the mitochondrial respiration by SA. It is reasonable to assume that the increase in Ca2+ level by e ATP could alleviate the SA-induced inhibition in respiration and iATP production, thus alleviating the cell death by SA.