Cadmium-induced subcellular accumulation of O2.- and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H2O2 and O-2(.-) production was studied in leaves from pea plants growth for 2 weeks with 50 muM Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl3 and Mn/DAB staining for H2O2 and O-2(.-), respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 muM CdCl2 a rise of six times in the H2O2 content took place in comparison with control plants, and the accumulation of H2O2 was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H2O2 was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H2O2 could be a NADPH oxidase. The subcellular localization of O-2(.-) production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd-induced production of the ROS, H2O2 and O-2(.-), could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd-grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca2+ channels, and cGMP.