Cadmium‐induced subcellular accumulation of O₂^·− and H₂O₂ in pea leaves

ABSTRACT Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H 2 O 2 and O 2 ·− production was studied in leaves from pea plants growth for 2 weeks with 50 µ m 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 CeCl 3 and Mn/DAB staining for H 2 O 2 and O 2 ·− , respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µ m CdCl 2 a rise of six times in the H 2 O 2 content took place in comparison with control plants, and the accumulation of H 2 O 2 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 H 2 O 2 was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H 2 O 2 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, H 2 O 2 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, Ca 2+ channels, and cGMP.