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

Abstract

SummaryA role for cytosolic pH (pHi) in hormonal signalling and transport control in plants has long been mooted. Yet, while changes in pHi 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, pHi 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 pHi 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 pHi values near 7.5, and evacuolation had no significant effect on this parameter. Reversible decreases of 0.1–0.2 units in pHi 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 pHi 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 pHi loads imposed by adding Na+‐butyrate to the bath. These results implicate the tonoplast or vacuolar compartment in short‐term pHi homeostasis and generation of hormonally evoked H+ signalling in plant cells; they also conform with the view that the decrease in pHiper se is not a primary determinant in the stimulation of H+ secretion by auxin.