In-situ determination of intracellular concentrations of K+ in barley (Hordeum vulgare L. cv. Kara) using the K+-binding fluorescent dye benzofuran isophthalate

Abstract

The tetra[acetoxymethyl] ester of the K+-binding fluorescent dye benzofuran isophthalate (PBFI-AM) was used to determine changes in intracellular potassium (K+) concentrations and to measure net transport of K+ in barley (Hordeum vulgare L. cv. Kara) root and leaf protoplasts. When this dye binds to free K+ inside the cytoplasm, the fluorescence intensity ratio 340/380 nm increases in direct relation to the K+ concentration. Because of a delay in the uptake of dye into the vacuoles, it is possible to determine K+ concentrations in the vacuoles and transport of K+ from the cytoplasm into the vacuole. The uptake of PBFI-AM in root and leaf protoplasts of barley differed in the absence or presence of external K+ and was faster at pH 5.5 than at pH 7.0. The fluorescence intensity of the dye was stable for at least 20 h when the protoplasts were kept at 4°C. In the presence of nigericin, the fluorescence intensity of both cells and protoplasts was linearly related to the external concentration of K+ (up to 100 mM).