Changes in inorganic phosphate (Pr) concentrations in barley leaves during growth of plants with .sufficient or deficient supplies of Pi were studied. Measurements of the Pi distribution from subcellnlar levels to the leaf tissue level under the same experimental conditions allowed us to analyse the relationship between the P, homeostasis of various compartments and Pj re-translocation in the whole plant. Under Pi deficiency, the finding of growth-dependent changes in the Pj concentrations of whole leaves established that Pi was re-translocated from the older leaves to the young leaves. Translocation of -"^Pi was also confirmed with an 'imaging plate' system, which made it possible to follow P; movement in the same plantlet. To analyse the mechanism of Pi re-translocation, the Pi distribution amongst various compartments of the leaves was measured. Under Pi deficiency, the cytoplasmic Pi concentration of the first leaf remained constant until 16d after sowing, while vacuolar Pi was completely exhausted after 8 to lOd. Exhaustion of vacuolar Pi in the first leaf coincided with the appearance of the second leaf. The Pi concentration in the apoplast changed similarly to that of the whole leaf. However, the apoplastic Pi concentration was aflected to some extent by the vacuolar Pi concentration and the growth of the younger leaf, because the main change in apoplastic Pi concentration coincided with the time of the disappearance of the vacuolar Pi and the appearance of the younger leaf. The Pi concentration in the apoplast was about 0 1 to 1 molm~\ even in the absence of Pi, which was much higher than that in the usual soil environment (a few mmolm^). This suggests that the Pi absorbed by root cells is concentrated in the transport process from the root to the leaf apoplast. The content of Pi in the xylem exudate was constant irrespective of growth culture conditions. The root may be functioning as the constant Pi supplier to the above tissues.

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 during inorganic phosphate (Pi) absorption by Catharanthus roseus cells were studied by means of a fluorescent pH indicator, 2',7'-bis-(2-carboxyethyl)-5 carboxyfluorescein (acetomethylester) (BCECF), and 31P-nuclear magnetic resonance spectroscopy. Cytoplasmic acidification measured by decrease in the fluorescence intensity started immediately after Pi application. Within a minute or so, a stable state was attained and no further acidification occurred, whereas Pi absorption was still proceeding. As soon as Pi in the medium was exhausted, cytoplasmic pH started to recover. Coincidentally, the medium pH started to recover toward the original acidic pH. The Pi-induced changes in the cytoplasmic pH were confirmed by 31P-nuclear magnetic resonance study. Maximum acidification of the cytoplasm induced by 1.7 millimolar Pi was 0.2 pH units. Vacuolar pH was also affected by Pi. In some experiments, but not all, pH decreased reversibly by 0.2 to 0.3 pH units during Pi absorption. Results suggest that the cytoplasmic pH is regulated by proton pumps in the plasma membrane and in the tonoplast. In addition, other mechanisms that could consume extra protons in the cytoplasm are suggested.