Determination of auxin-dependent pH changes in coleoptile cell walls by a null-point method

The present debate on the validity of the "acid-growth theory" of auxin (indole-3-acetic acid, IAA) action concentrates on the question of whether IAA-induced proton excretion into the cell wall is quantitatively sufficient to provide the shift in pH that is required to explain IAA-induced growth (see D.L. Rayle, R.E. Cleland [1992] Plant Physiol 99:1271-1274 for a recent apologetic review of the acid-growth theory). In the present paper a null-point method has been employed for determining the growth-effective cell-wall pH in the presence and absence of IAA after 60 min of treatment. Elongation of abraded maize (Zea mays L.) and oat (Avena sativa L.) coleoptile segments was measured with the high resolution of a displacement transducer. The abrasion method employed for rendering the outer epidermal cell wall permeable for buffer ions was checked with a dye-uptake method. Evidence is provided demonstrating that externally applied solutes rapidly and homogeneously penetrate into the epidermal wall, whereas penetration into the inner tissue walls is strongly retarded. "Titration" curves of IAA-induced and basal elongation were determined by measuring the promoting/inhibiting effect of medium pH under iso-osmotic conditions in the range of pH 4.5 to 6.0. In maize, the null point (no pH-dependent change in elongation rate after 5-10 min of treatment with 10 mmol L(-1) citrate buffer) was pH 5.00 after 60 min of IAA-induced growth, and the null-point pH determined similarly in IAA-depleted tissue (10 times smaller elongation rate) was 5.25. Corresponding titration curves with Avena segments led to slightly lower null-point pH values both in the presence and absence of IAA-induced growth. After induction of acid-mediated extension by 1 micromole L(-1) fusicoccin (FC) in maize, the null-point pH shifted to 3.9. At 0.5 micromole L(-1), FC induced the same elongation rate as IAA but a 9-fold larger rate of proton excretion. At 0.033 micromole L(-1), FC induced the same rate of proton excretion as IAA but had no appreciable effect on elongation. The implications of these results against the background of recent attempts to revitalize the acid-growth theory of IAA action are discussed.