Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured Cells of Catharanthus roseus (L.) G. Don

Abstract

Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don caused a rapid alkalinization of the medium in which they were suspended. The alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the pH of the medium started to drop sharply toward the original pH value. Phosphate exposure caused the pH of the medium to increase from pH 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10-17 micromoles per hour per gram fresh weight). This indicates that no apparent pH optimum exists for the phosphate uptake by the cultured cells. The amount of protons cotransported with phosphate was calculated from the observed pH change up to the maximum alkalinization and the titration curve of the cell suspension. Proton/phosphate transport stoichiometry ranged from less than unity to 4 according to the amount of phosphate applied. At low phosphate doses, the stoichiometries were close to 4, while at high phosphate doses, smaller stoichiometries were observed. This suggests that, at high phosphate doses, activation of the proton pump is induced by the longer lasting proton influx acidifying the cytoplasm. The increased H+ efflux due to the proton pump could partially compensate protons taken up via the proton-phosphate cotransport system. Thus, the H+/H2PO4-stoichiometry of the cotransport is most likely to be 4. MATERIALS AND METHODS Plant MaterialsCultured cells of Cathananthus roseus were obtained from the stock cultures of Dr. Ashihara of Ochanomizu University, and cultured in Murashige and Skoog basal medium (8) supplemented with 4.5 Amol of 2,4-D and 3% sucrose as described previously (1 1). Cells cultured for 6 to 10 d after transfer to fresh medium were collected on a filter paper under suction and washed with 10 mM CaCl2. Cells were weighed and suspended at the density of 5 g fresh weight in 50 mL of incubation medium consisting of 10 mm CaCl2, 10 mM KCI, and 1% glucose in a 100 mL Erlenmeyer flask. The flask was stoppered with a sleeve-ventilating plastic cap to which a glass pH electrode (TOA Electronics, GST-

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Cytoplasmic Acidification Induced by Inorganic Phosphate Uptake in Suspension Cultured Catharanthus roseus Cells

“…At the same time, when the cells were suspended in unbuffered medium, we could follow the H+/Pi cotransport by measuring medium pH (15) by a miniature glass pH electrode well. Figure 2 shows Pi-induced changes in the pH of unbuffered medium and in fluorescence intensity of the cells.…”

“…For the addition of Pi into a cuvette, a 10-mM solution of Na2HPO4 with a pH nearest to the medium pH (at the addition) was chosen from a series of solutions with pH ranging from 3.0 to 7.0 (pH interval 0.5, ref. 15).…”

“…In the fluorescence measurements in unbuffered medium, phosphate uptake could be followed by monitoring changes in the medium pH; upon Pi addition, the medium pH continued to increase until the added phosphate was completely absorbed by the cells (15).…”

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