Vacuolar chloride transport in Mesembryanthemum crystallinum L. measured using the fluorescent dye lucigenin

To study vacuolar chloride (C1(-)) transport in the halophilic plant Mesemb ryanthemum crystallinum L., C1- uptake into isolated tonoplast vesicles was measured using the Cl--sensitive fluorescent dye lucigenin (N,N'-dimethyl- 9,9'-bisacridinium dinitrate). Lucigenin was used at excitation and emissio n wavelengths of 433 nm and 506 nm, respectively, and showed a high sensiti vity towards C1-. with a Stern-Volmer constant of 173 M-1 in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluo rescence intensity and Cl--sensitivity of lucigenin was shown to be strongl y affected by alkaline pH and was dependent on the conjugate base used as t he buffering ion. Chloride transport into tonoplast vesicles of M. crystall inum loaded with 10 mM lucigenin showed saturation-type kinetics with an ap parent K-m of 17.2 mM and a V-max of 4.8 mM min(-1). Vacuolar Cl- transport was not affected by sulfate, malate, or nitrate. In the presence of 250 mu M p-chlolomelcuribenzene sulfonate, a known anion-transport inhibitor, vac uolar C1- transport was actually significantly increased by 24%. To determi ne absolute fluxes of C1- using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1 .13 x 10(7) m(-1). After correcting for a 4.4-fold lower apparent Stern-Vol mer constant for intravesicular lucigenin, a maximum rate of Cl- transport of 31 nmol m(-2) sec(-1) was calculated, in good agreement with values obta ined for the plant vacuolar membrane using other techniques.