Generation of nitric oxide and hydrogen peroxide during the isolation and culture of Brassica napus leaf protoplasts

Abstract

The isolation of protoplasts is a stress-inducing process, because the tissue is treated with an enzyme solution containing pectinase, cellulase, and sorbitol. The enzyme solution used for protoplast isolation induced H₂O₂ and NO accumulation in chloroplasts. The levels of H₂O₂ and NO were highest just after protoplast isolation and subsequently decreased during culture. NADPH oxidase (NOX)-like activity was upregulated, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities were decreased in the chloroplasts compared with protoplasts and leaves. Guaiacol peroxidase was not detected in B.napus leaves, protoplasts, or chloroplasts. These results indicate that NOX-like activity in the chloroplasts generated excessive O₂^-, which subsequently dismutated to H₂O₂. A flow cytometric examination of protoplasts revealed lower mitochondrial membrane potential along with increased nuclear DNA fragmentation during protoplast culture. These results indicate that NO and ROS generated in the chloroplasts induced apoptotic cell death in B.napus leaf protoplasts.