OSMOTIC-STRESS ENHANCES THE COMPETENCE OF BETA-VULGARIS VACUOLES TO RESPOND TO INOSITOL 1,4,5-TRISPHOSPHATE

Isolated vacuoles from Beta vulgaris storage roots respond to the intracellular signalling molecule inositol 1,4,5-trisphosphate (InsP(3)). Whole vacuole patch clamp enables measurement of an inward current (cytosol-directed) induced by cytosolic InsP(3) which is fully reversible upon removal of InsP(3). The reversal potentials of the InsP(3)-induced whole vacuolar currents indicate a permeability ratio (P-Ca:P-K) of 200:1. Competence of vacuoles to respond to InsP(3) is dependent upon the root tissue undergoing hyperosmotic stress before vacuole isolation. The magnitude of the hyperosmotic stress and the density of InsP(3)-induced current per unit membrane area are exponentially related. A standing osmotic gradient across the vacuolar membrane further enhances the InsP(3)-induced current, the current being larger when there is net water flux from the cytosol to the vacuolar lumen. InsP(3)-induced currents are not affected by the cytosolic free Ca2+ concentration. The conductance of InsP(3)-induced single channel currents varied greatly between individual outside-out patches, but all showed a non-linear increase in single channel current at physiological potentials. The reversal potentials of these currents indicated a P-Ca:P-K of between 100:1 and 800:1. The significance of these findings is discussed in relation to technical aspects of monitoring InsP(3)-induced currents in plant vacuoles and in the context of the physiological roles of InsP(3) and its receptor in cell water relations.