A role for the vacuole in auxin‐mediated control of cytosolic pH by Vicia mesophyll and guard cells

A role for cytosolic pH (pH_i) in hormonal signalling and transport control in plants has long been mooted. Yet, while changes in pH_i are a common consequence of hormonal stimuli in plant cells and contribute to hormonally evoked ion channel control, the origins of these changes remain unknown. To examine a possible role for the tonoplast and vacuolar compartment in these events, pH_i was measured in the presence of auxins and during cytosolic H⁺ loading with weak acid in vacuolate and evacuolate protoplasts, both from mesophyll and guard cells of Vicia faba L. Evacuolate protoplasts were obtained following ultracentrifugation on Percoll gradients, and pH_i of single protoplasts was recorded in both vacuolate and evacuolate preparations using fluorescence ratio microphotometry and the pH-sensitive dye BCECF. External pH measurements indicated a roughly twofold increase in the rate of net H⁺ secretion in evacuolate compared with vacuolate protoplasts, and showed that evacuolate protoplasts retained the characteristic stimulation of H⁺ secretion in the presence of auxin. BCECF fluorescence recording gave resting pH_i values near 7.5, and evacuolation had no significant effect on this parameter. Reversible decreases of 0.1–0.2 units in pH_i were evoked in vacuolate protoplasts by 10 μM concentrations of the auxins 1-naphthalene acetic acid and 3-indoyl-acetic acid, and not by the inactive (anti-auxin) analogue 2-naphthalene-acetic acid. However, auxin treatments failed to evoke a change in pH_i in all but one of 12 experiments with evacuolate protoplasts. Evacuolation also appeared to reduce the transient, dynamic H⁺ buffering capacity of the protoplasts in the face of acid pH_i loads imposed by adding Na⁺-butyrate to the bath. These results implicate the tonoplast or vacuolar compartment in short-term pH_i homeostasis and generation of hormonally evoked H⁺ signalling in plant cells; they also conform with the view that the decrease in pH_iper se is not a primary determinant in the stimulation of H⁺ secretion by auxin.