Phosphate uptake across the tonoplast of intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don

Transport of inorganic orthophosphate (Pi) across the tonoplast membrane wa s studied using intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus. Orthophosphate uptake was strongly stimulated in the p resence of Mg-ATP and Mg-pyrophosphate and inhibited by bafilomycin and con canamycin which are potent inhibitors of the vacuolar H+-ATPase. These resu lts indicated that the buildup of an electrochemical gradient by the H+ pum ps tvas essential for the uptake of Pi. Potassium thiocyanate, which dissip ates the membrane potential across the tonoplast, strongly inhibited the Mg -ATP-stimulated uptake of Pi, while only a weak inhibition was observed in the presence of NH4Cl, which dissipates the pH gl-adient. These results ind icate that, as observed for other anions like malate or chloride, the elect rical component is the driving force of Pi uptake, whereas the Delta pH pla ys only a minor role. Possible competitive inhibitors of Pi, MoO42-, VO43- and CrO42- were tested. Among them, CrO42- strongly inhibited Pi uptake int o the vacuoles. Various inhibitors of anion transport were also tested. Onl y 4,4-diisothiocyanostilbene-2,2'-disulfonic acid strongly inhibited Pi upt ake into the vacuoles. The function of the vacuolar Pi transporters for cyt oplasmic Pi homeostasis is discussed.