Effects of superoxide radicals on ACC synthase activity in chilling-stressed etiolated mungbean seedlings

The activity of 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase, ACS) and the concentrations of superoxide radical (O₂^−.) and hydrogen peroxide (H₂O₂) were measured in etiolated mungbean seedlings following their transfer to a growth chamber at 25°C after a 5-h-chilling treatment at 5°C. All of these variables increased dramatically after the transfer, and strong correlations were found between ACS activity and the concentrations of superoxide and H₂O₂. Exogenous applications of two generators of superoxide radicals, methylviologen (MV) and xanthine–xanthine oxidase (X–XOD), enhanced ACS activity in seedlings, but their effects were inhibited by exogenous applications of specific scavengers of O₂^−.. However, applications of H₂O₂ or specific H₂O₂-scavengers had no significant effects on seedlings ACS activity. The results indicate that O₂^−. was involved in the chilling-induced increases in ACS activity, but not H₂O₂. ACS activity peaked ca. 8 h after the transfer, and then declined, but the decline could be counteracted by exogenous applications of specific O₂^−. scavengers, this suggests that damage was caused by superoxide radicals influencing ACS activity in etiolated mungbean seedlings. Further analysis of changes in two key kinetic parameters of ACS activity—V _max (maximum velocity) and K _m (the Michaelis constant)—in the seedlings indicated that the presence of O₂^−. may reduce K _m, i.e. increase substrate (S-adenosyl methionine, SAM) affinity. That would be the main mechanism responsible for the observed chilling-induced increases in ACS activity in etiolated mungbean seedlings.