Cell Death Induced By CuCl₂Stress In Tobacco BY-2Cells

The key factors for cell death induced by stress conditions have remained unclear.Moreover, the mechanisms of the cell death induced by CuCl₂stress and the effect of CuCl₂stress on the mitochondrial respiration and the relationship between the mitochondrialrespiration and cell death have not yet been known. Tobacco BY-2cell is a cell line withoutfunctional chloroplasts. The BY-2cell was used in this study to explore the mechanism of thecell death induced by CuCl₂stress without influence of reactive oxygen species （ROS）mediated by photosynthetic electron transport and to explore the role of mitochondrialinhibitory processes in the cell death of tobacco BY-2cells.The results showed that:0.5mmol·L^-1CuCl₂led to remarkable cell death, the opening ofmitochondrial permeability transition pore （MPTP）, the release of cytochrome c （Cyt c） frommitochondrial to cytoplasm in tobacco BY-2cells. The degree of cell growth inhibition, thedamage of cell membrane and the rate of cell death increased with the CuCl₂treatment time.What’s more, the CuCl₂treatment resulted in overproduction of reactive oxygen species（ROS）, a rapid depletion of ATP and a decline of respiration in tobacco BY-2cells.Furthermore, it was demonstrated that the direct inhibition of mitochondrial electron transportchain （ETC）, alternative oxidase （AOX） pathway capacity, cytochrome pathway capacity andCAT activity by CuCl₂treatment might be the main contributors to the CuCl₂-induced ROSburst in tobacco BY-2cells. These results indicate that the inhibition of the respiration and thecytochrome pathway inevitably resulted in ATP depletion in tobacco BY-2cells. Theuncoupling of the oxidative phosphorylation induced by CuCl₂was another contributor to theATP depletion, and the inhibition of the AOX pathway by the CuCl₂treatment was one of themain reasons which led to the ROS burst in tobacco BY-2cells.When the cells were treated with the CuCl₂plus CAT, ROS overproduction was blocked,however, the cell deathwas not prevented yet. When CAT was added to the suspension1hbefore CuCl₂treatment, the cell death was prevented in the CuCl₂treated BY-2cells.Moreover, the addition of exogenous adenosine together with CuCl₂significantly inhibitedATP depletion and prevented cell death. The data presented here demonstrate that the ATPdepletion played an important role in CuCl₂-induced cell death in the tobacco BY-2cells.However, the addition of exogenous adenosine not only suppressed ATP depletion, but it mayalso block ROS overproduction induced by CuCl₂stress. The data obtained in this study are not enough to prove that ATP depletion is the immediate cause of cell death induced by theCuCl₂treatment. It needs a lot of further studies to address the precise role of extracellularATP depletion in cell death, because ATP is a ubiquitous energy source that can also act as asignaling molecule in cellular metabolism.