Excessive copper induces the production of reactive oxygen species, which is mediated by phospholipase D, nicotinamide adenine dinucleotide phosphate oxidase and antioxidant systems.
Tobacco BY-2 suspension cells were used to study the chemical damage and its associated mechanisms caused by Cu2+. Treatment with 100 micromol/L Cu2+ generated a large amount of H2O2 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) oxidase-overexpressing 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 Cu2+. Moreover, the content of ascorbic acid (AsA), an effective antioxidant, was sharply reduced in BY-2 cells exposed to excessive Cu2+. Furthermore, a significant downregulation of the enzymes of AsA biosynthesis and the antioxidant system was found. This evidence suggests that excessive Cu2+-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.
Yu
Zhong-Lian
ZL
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China.
Zhang
Jin-Guang
JG
Wang
Xue-Chen
XC
Chen
Jia
J
D000975:Antioxidants / Q000378:metabolism*; D001205:Ascorbic Acid / Q000378:metabolism; D003300:Copper / Q000633:toxicity*; D003470:Culture Media; D004789:Enzyme Activation / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008954:Models, Biological; D019255:NADPH Oxidases / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D017382:Reactive Oxygen Species / Q000378:metabolism*; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
D000975:Antioxidants; D003470:Culture Media; D010712:Phosphatidic Acids; D010940:Plant Proteins; D017382:Reactive Oxygen Species; D017392:Thiobarbituric Acid Reactive Substances; D003300:Copper; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D010739:Phospholipase D; D001205:Ascorbic Acid