Ethylene- and enzyme-mediated superoxide production and cell death in carrot cells grown under carbon starvation

Carrot (Daucus carota L.) suspension cells are known to exhibit acclimation responses, such as fatty acid catabolism, during the first 5 days of carbon starvation. The present study shows that cells start to die once starvation is prolonged beyond this period. We investigated the signals involved in this transition from acclimation to cell death. Significant amounts of superoxide were produced transiently at days 4-5 (after the initiation of starvation). When diphenylene iodonium, an inhibitor of NADPH oxidase and/or other ROS (reactive oxygen species)-generating enzymes, was added to the cell suspension at the beginning of the starvation period, superoxide production and cell death, as measured by internucleosomal cleavage, were effectively inhibited, indicating that NADPH oxidase and/or other ROS-generating enzymes are involved in the production of the superoxide during starvation. Ethylene, which is known to be produced transiently at day 1 in these cells, positively regulated production of the superoxide and cell death, indicating that ethylene is an upstream regulator of NADPH oxidase or other ROS-generating enzymes. We suggest that as the carrot cells are dying under conditions of carbon starvation they put the following signal relays into operation: ethylene production --> activation of NADPH oxidase (or other ROS-generating enzymes) --> superoxide production --> internucleosomal cleavage (cell death).