The effects of excess copper (Cu) on the production of reactive oxygen species (ROS) and antioxidant enzyme activities in leaves of the Cu-accumulator Elsholtzia haichowensis Sun were investigated. The addition of 100 mu M of copper significantly increased the accumulation of hydrogen peroxide (H2O2) and the activities of superoxide dismutase (SOD) and other antioxidant enzymes. The increase in SOD activity was attributable to an increase in apoplastic and symplastic copper-zinc superoxide dismutase (CuZn-SOD) activity. Induction of CuZn-SOD proteins was demonstrated by immunoblot analysis. This study also provides the first cytochemical evidence of an accumulation of superoxide anion in the chloroplasts of mesophyll cells, and H2O2 in the mesophyll cell walls and extracellular space, as a consequence of Cu treatment. Experiments with diphenyleneiodonium as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and N-N-diethyldithiocarbamate as an inhibitor of SOD showed that the source of H2O2 in the cell wall could be, in part, NADPH oxidase. Apoplastic guaiacol peroxidase (POD) and symplastic ascorbate peroxidase (APX) activities were induced in leaves of E. haichowensis with 100 mu M Cu, suggesting that apoplastic POD and symplastic APX may be important in avoiding the buildup of toxic H2O2 concentrations. (C) 2010 Elsevier B.V. All rights reserved.