Effects of Bafilomycin A1 and Metabolic Inhibitors on the Maintenance of Vacuolar Acidity in Maize Root Hair Cells

Proton pumps of tonoplast membranes have been studied extensively in vitro, but data concerning their regulation in vivo are lacking. Effects of either anoxia, or the addition of KCN, 2-deoxy-d-glucose (deoxy-glucose), or bafilomycin-A1 (BAF) on vacuolar pH of maize (Zea mays L.) root hair cells were followed by fluorescence microscopy after loading of 2[prime]7[prime]-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein. Root hair cells were able to maintain vacuolar acidity for at least 2 h in the presence of either 10 mM KCN or 50 mM deoxy-glucose or during anoxia. Treatments with either deoxy-glucose or KCN reduced total tissue ATP more than anoxia. ADP accumulated during anoxia and treatment with KCN as detected by in vivo 31P-NMR spectroscopy, but not during deoxy-glucose treatment. With control roots and roots treated with deoxy-glucose, the presence of BAF, a specific inhibitor of the V-type ATPase, caused alkalization of the vacuolar pH. However, either in the presence of KCN or under anoxic conditions, BAF was relatively ineffective in dissipating vacuolar acidity. Therefore, under anoxia or in the presence of KCN, unlike the situation with air or deoxy-glucose, the V-type ATPase apparently is not required for maintenance of vacuolar acidity.