Diphenylamine Inhibits Respiration of Green Bell Peppers

The mechanism by which diphenylamine (DPA) controls superficial scald in apples and reduces chilling injury in green bell peppers (Capsicum annuum L. var. annuum (Grossum Group)) has been assumed to be related to its antioxidant activity. In the present study, DPA inhibited the respiratory activity of green bell pepper fruit as well as oxygen uptake by the mitochondria isolated from them. When the alternative oxidase was inhibited with n-propyl gallate or disulfiram during state 4 respiration, DPA did not further inhibit O2 uptake. Treating green bell peppers with DPA before storage did not alter the induction and abundance of the alternative oxidase protein in mitochondria which was maximally induced in peppers stored at 4 °C. Whether added before or after the uncoupler, 2,4-dinitrophenol, DPA negated the enhanced O2 uptake associated with uncoupling of electron transfer in isolated mitochondria. These observations indicate that DPA inhibits the flow of electrons through the cytochrome path, probably somewhere in the cytochrome bc1 complex. Although the secondary amine function of DPA makes it a powerful antioxidant, the effectiveness of DPA in reducing chilling injury in green bell peppers and superficial scald in apples (Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf. ) also may be due, in part, to its inhibition of respiration. Diphenylamine (DPA), used commercially in the United States to control low-temperatureinduced superficial scald in apples (Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf. ) during storage (Smock, 1961), was recently reported to reduce chilling injury in cold-stored green bell pepper (Capsicum annuum L. var. annuum (Grossum Group)) fruit (Purvis, 2002). The mechanism by which DPA reduces chilling injury in bell pepper and superficial scald in apple is not known, but the prevalent hypothesis for the reduction of superficial scald in apples is that DPA inhibits the oxidation of _ -farnesene to conjugated trienes, which are thought to be toxic to fruit tissues, possibly through their initiation of free-radical-me- diated chain reactions and/or decomposition to harmful volatiles (Whitaker, 2000). Diphenylamine, however, also reduced _ -farne- sene synthesis, ethylene production and respiration in apples (Lurie, et al., 1989; Whitaker, 2000), and altered chlorophyll fluorescence parameters in cold stored green bell peppers (Purvis, 2002), but had no effect on chlorophyll fluorescence parameters in apples stored in refrigerated air (Mir et al., 1998). Diphenylamine was first tested as a potential inhibitor of apple scald (Smock, 1955, 1961) based on a report that it inhibited cyto- chrome oxidase activity in mitochondria isolated from rabbit liver (Abood and Gerard, 1953). Cytochrome oxidase is the terminal oxidase in the electron transport chain of both animal and plant mitochondria. Diphenylamine was found to only marginally inhibit cytochrome oxidase activity in mitochondrial particles of apple fruit but it severely inhibited the oxidation of succinate (Yatsu, 1960). However, succinate dehydrogenase and NADH diaphorase (dehydrogenase) activities of sweetpotato (Ipomoea batatas L.) mitochondria were not inhibited by DPA (Baker, 1963). Since the reduction of the cytochromes was delayed, but no reduced-oxidized pairs were observed, Baker (1963) suggested that DPA inhibits