Generation of intracellular reactive oxygen species during the isolation of Brassica napus leaf protoplasts

The isolation of leaf protoplasts is a stress-inducing procedure that generates reactive oxygen species (ROS). We found that a cellulase-pectinase enzyme solution used for isolating protoplasts contained peroxidase (POX), catalase (CAT), and superoxide dismutase (SOD) activity, and effectively scavenged externally added hydrogen peroxides. During the isolation of Brassica napus leaf protoplasts, the protoplasts accumulated 1.5 times more intracellular H2O2 than normal leaf cells, but the H2O2 released into the medium increased only negligibly during culture. H2DCF-DA and DAF2-DA were used as probes for the intracellular localization of H2O2 and NO with a confocal laser scanning system. H2O2 and NO had different intracellular localizations in freshly prepared B. napus leaf protoplasts. These results indicate that intracellular ROS are generated by the stresses of protoplast isolation and will induce the apoptosis-like cell death of the cultured protoplasts.