Tobacco BY-2 suspension cells were used to study the chemical damage and its associated mechanisms caused by Cu^2＋. Treatment with 100 μmol/L Cu^2＋ generated a large amount of HzOz and thiobarbituric acid-reactive substances （TBARS） in cells. Using phospholipase D （PLD） specific inhibitor （1-butanol） or phosphatidic acid （PA）, we demonstrated that PLD plays an important role in the generation of H2O2 and TBARS. Semi-quantitative reverse-transcriptase polymerase chain reaction and enzyme activity assays with wild type and nicotinamide adenine dinucleotide phosphate （NADPH） oxidaseoverexpressing BY-2 cells revealed that PLD and PA are the key factors leading to NADPH oxidase activation, which is responsible for H2O2 and TBARS production induced by Cu^2＋. Moreover, the content of ascorbic acid （AsA）, an effective antioxidant, was sharply reduced in BY-2 cells exposed to excessive Cu^2＋. Furthermore, a significant downregulation of the enzymes of AsA biosynthesis and the antioxidant system was found. This evidence suggests that excessive Cu^2＋-elevated reactive oxygen species （ROS） production is caused by upregulated PLD that elevates the activity of NADPH oxidase and its collapsed antioxidant systems that scavenges ROS.
Zhong-Lian Yu Jin-Guang Zhang Xue-Chen Wang Jia Chen
