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 c ontrol in plants has long been mooted. Yet, while changes in pH(i) are a common consequence of hormonal stimuli in plant cells and contribut e to hormonally evoked ion channel control, the origins of these chang es remain unknown. To examine a possible role for the tonoplast and va cuolar 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 Vi cia faba L. Evacuolate protoplasts were obtained following ultracentri fugation on Percoll gradients, and pH(i) of single protoplasts was rec orded in both vacuolate and evacuolate! preparations using fluorescenc e ratio microphotometry and the pH-sensitive dye BCECF. External pH me asurements indicated a roughly twofold increase in the rate of net Hsecretion in evacuolate compared with vacuolate protoplasts, and showe d 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 significa nt effect on this parameter. Reversible decreases of 0.1-0.2 units in pH(i) were evoked in vacuolate protoplasts by 10 mu M concentrations o f the auxins 1-naphthalene acetic acid and 3-indoyl-acetic acid, and n ot by the inactive (anti-auxin) analogue 2-naphthalene-acetic acid. Ho wever, auxin treatments failed to evoke a change in pH(i) in all but o ne of 12 experiments with evacuolate protoplasts. Evacuolation also ap peared to reduce the transient, dynamic H+ buffering capacity of the p rotoplasts in the face of acid pH(i) loads imposed by adding Na+-butyr ate to the bath. These results implicate the tonoplast or vacuolar com partment in short-term pH(i) homeostasis and generation of hormonally evoked H+ signalling in plant cells; they also conform with the view t hat the decrease in pH(i) per se is not a primary determinant in the s timulation of H+ secretion by auxin.