A hydrogen ion-selective liquid-membrane microelectrode for measurement of the vacuolar pH of plant cells in suspension culture

Abstract

H +-selective microelectrodes were made according to the method described by D. Ammann, F. Lanter, R. A. Steiner, P. Schulthess, Y. Shijo, and W. Simon (1981, Anal. Chem. 53, 2267–2269). Some practical aspects of the preparation and use of these microelectrodes for in vitro and in vivo pH measurements in plant vacuoles have been examined. The trials indicate that the microelectrodes can be kept for up to 48 h without modification of their slope (mV/pH unit) and resistance. The H +-selective liquid can be used more than 4 months after being prepared. The vacuole is known to be the storage compartment of plant cells where solutes are accumulated; as an example, sucrose and a model protein, bovine serum albumin (BSA), were chosen to study the effect of solutes on the response of the microelectrodes. The results indicate that the slope of the regression line is not modified by sucrose (20–100 m m) added to citrate buffer solution, but it is slightly decreased when the microelectrodes are tested in the presence of BSA or in plant juice, indicating that some components of the cell sap are able to interfere with and to modify the response of the microelectrodes. More experiments are needed in order to determini it proteins, ions, or another substance are the factors causing this effect. Microelectrodes with tip diameters in the range 0.3–0.6 μm and electrical resistance in the range 2 × 10 12 ohms are shown to be suitable for the measurement of vacuolar pH in plant cells. Their short response time (several seconds) when inserted into vacuoles makes them appropriate for follwing vacuolar pH modifications in situ.