Generation of intracellular reactive oxygen species during the isolation of Brassica napus leaf protoplasts
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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 <i>Brassica napus</i> leaf protoplasts, the protoplasts accumulated 1.5 times more intracellular H<sub>2</sub>O<sub>2</sub> than normal leaf cells, but the H<sub>2</sub>O<sub>2</sub> released into the medium increased only negligibly during culture. H<sub>2</sub>DCF-DA and DAF2-DA were used as probes for the intracellular localization of H<sub>2</sub>O<sub>2</sub> and NO with a confocal laser scanning system. H<sub>2</sub>O<sub>2</sub> and NO had different intracellular localizations in freshly prepared <i>B. napus</i> 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.
- Plant tissue culture letters
Plant tissue culture letters 24(4), 361-366, 2007-09-01
Japanese Society for Plant Cell and Molecular Biology