Over-reduction of cultured tobacco cells mediates changes in respiratory activities

Regulation of respiration in Nicotiana tabacum suspension cultures was studied using the respiratory inhibitor myxothiazol and the oxidative phosphorylation uncoupler carbonylcyanide p( trifluoromethoxy) phenylhydrazone (FCCP). Myxothiazol (2 muM) or FCCP (2 muM) almost completely inhibited cell growth for about 24 h, after which the cultures could resume a growth rate similar to that of the untreated culture. During the first 18 h of myxothiazol treatment, rates of cellular respiration were similar to control cultures. The capacity of the alternative pathway was significantly higher than control 2-3 h after myxothiazol treatment and increased further after 18 h. The capacity of the alternative pathway in FCCP-treated tobacco cells also increased but to a lesser extent. ATP/ADP ratios decreased for at least 9 h after either myxothiazol or FCCP treatments, which triggered an increase in glycolytic flux and an over-reduction of cultured tobacco cells, as demonstrated by increases in levels of reactive oxygen species and cellular ethanol. The generation of reactive oxygen species served as a signal for an induction of alternative oxidase, which in turn relieved the aerobic fermentation and over-reduction of the cells.