Previous workl-5 has shown the existence of a high-energy condition of isolated chloroplasts, caused by illumination in the absence of phosphate or ADP. This state is inferred from the ability to form ATP in the postillumination darkness. Accompanying the condition is an apparent uptake of hydrogen ions6z I together with an excretion of Mg++ or other cations.8 A feasible interpretation, stemming from the "chemi-osmotic" hypothesis for the mechanism of phosphorylation in double membrane containing organelles,9' 10 is that illumination causes the uptake of hydrogen ions into the inner space of the grana disk double membranes. The resulting inequality in hydrogen ion electrochemical activity across the membrane is postulated to be, in itself, the high-energy condition, able to drive the formation of ATP. If this interpretation is correct, then the same high-energy condition should be formed artificially, entirely in the dark and without electron transport, by loading the inner space of the grana disk membranes with protons. If this could be accomplished by placing chloroplasts in an acid medium, when first returned to pH 8 they might be expected to make some ATP due to the pH gradient across the membranes. The operational formation of ATP in this manner, entirely in the dark, was discovered and noted briefly earlier.4 The present paper represents an extension and further exploration of this phenomenon of "acid-bath" dark phosphorylation by chloroplasts. Materiats and Methods.-Chloroplasts were prepared from market spinach as described pre

ABSTRACTof photosynthesis observed at above-optimum temperatures (3).Temperature has also been shown to affect both the Km (CO2) and V,= of RuBP carboxylase in vitro (2,24). Weis (35) has suggested that a temperature-dependent inactivation of RuBP carboxylase occurs at above-optimal temperatures in intact spinach chloroplasts. Such temperature effects may be partly responsible for the reversible temperature limitations on net photosynthesis observed for intact leaves. However, difficulties arise in interpreting these data due to the uncertainties encountered in using in vitro studies to describe processes in vivo.Photosynthetic inhibition at analysis temperatures beyond the point of reversibility is due in part to irreversible inhibition of the quantum yield for CO2 fixation, and decreased activity of certain enzyme reactions (4). An irreversible reduction in the quantum yield has been used as an indication of heat damage to the photosynthetic apparatus (29), and is presumably related to the integrity of the thylakoid membranes (1).Agropyron smithii is a C3 perennial grass which constitutes a major biomass producer of the short-and mixed-grass prairie ecosystems. Plants of this species initiate growth during the early spring months when seasonal temperatures are relatively cool (6). (P2%0-P21%0,/P2%0,) 100(1)Leaf transmittance and reflectance measurements were conducted on freshly cut leaf discs, at 5-nm wavelength intervals between 400 and 700 nm, with the integrating sphere described by Robberecht and Caldwell (28 min prior to initiating each assay, a 2-to 3-ml aliquot of the frozen extract was removed from the mortar and allowed to thaw at room temperature. The thawed extract was pressed through a 40-,um nylon net attached to a 20-ml syringe and irnmediately assayed.The assays were conducted in small glass vials which were half immersed in a temperature-controlled water bath. The assay buffer (100 mM C02-free Tricine, pH 8.0), which contained 5 mm DTT and 20 mm MgCl2, was added to each reaction vial approximately 2 min prior to each assay in order to insure temperature equilibration. N2 was bubbled through the assay medium for the entire 2-min equilibration period. Carbonic anhydrase (100 units) and RuBP (0.4 mm) were added to the assay medium at 60 and 45 s prior to initiating each assay, respectively. The appropriate concentration of NaH4C03-(7.0 mCi mm-) was added 5 s before initiating the reaction, thus minimizing losses of 14CO2 to the atmosphere above the reaction medium. The reaction was initiated by the addition of 20 IAl of crude extract. The final volume of the reaction mixture was 1 ml. The reaction was terminated after 30 s (1 min at 10 and 15°C) by the addition of 200 ,ul of 6 N HCI. The contents of each reaction vial were transferred to a scintillation

The effect of temperature on respiration and kinetics of H2PO4-and K+ uptake in corn roots was determined in the range of 2 to 42 C. The response of uptake to temperature, determined from Qio and activation energy (Ea) data, for the anion and the cation differ significantly, especially in the range of uptake mechanism (Mech.) I. At 2.5 micromolar the Ea for K+ uptake below the 13 C transition is 29.3 kilocalories per mole. As the K+ concentration is increased, Ea (22.7 at 1.0 millimolar to 1.0 at 50 millimolar). There is no definable low temperature transition at these concentrations. Ion uptake is found to be much more sensitive to low temperature than respiration in this chillsensitive species. The data suggest that the low temperature reduction of ion transport is more closely related to restriction of function of active transport systems than to either respiration or membrane permeability.An analysis of the relationship of temperature to the polarization of the cell PD4 and respiration of corn roots revealed that oxidative metabolism is not a limiting factor for ion uptake below the apparent membrane transition of corn roots (2). Low temperature inhibition of the ion uptake capabilities of chill sensitive species is well documented (5, 6) and several hypotheses involving membrane localized effects have been advanced to explain this phenomenon (5). The extreme temperature dependence of the membrane potential below the apparent phase transition and the marked decline above it suggested that a major effect of low ' Partially supported by a grant from the Universidad Central de MATERIALS AND METHODS Seedlings of corn (Zea mays L. cv. Golden Bantam) were grown as described in the accompanying paper. After the 48-h period in lx Higinbotham solution (11) 5-cm apical root sections were excised and rapidly transferred to lx solution. These were recut 2.5 cm behind the tip just prior to use and rinsed in deionized H20. Approximately 0.5 g of root segments were placed in culture tubes which contained 50 ml of vigorously aerated absorption solution.The uptake solution for the Pi uptake experiments, unless otherwise specified, was composed of 0.25 mm KH2PO4 buffer plus 0.20 mm CaCl2 at pH 6.0. For the K+ uptake experiments, the absorption solution unless otherwise specified, was composed of 0.25 mm KCI plus 0.20 mm CaCl2 at £H 5.5. Absorption solutions were labeled with 5 ,uCi/l of 32p or Rb, respectively. At the end of the absorption period, which was I h except for the time courses of Pi uptake, the root segments were rinsed for 30 s with two 10-ml aliquots of unlabeled exchange solution. This solution was composed of 5 mm KH2PO4 plus 0.20 mm CaCl2 at pH 6.0 for Pi exchange, and 1.0 mm KCI plus 0.20 mm CaCl2 at pH 5.5 for K+ exchange. Roots were then transferred to ice-cold unlabeled exchange solution for removal of ions from the free space, as described by Epstein et al. (9). Exchange was 30 min for K+ and 10 min for Pi. Apparent ion uptake was determined by liquid scintillation spectrometry, using a Pack...

The brown algae Desmarestia ligulata var. ligulata (Lightf.) Certain species of the macrophytic brown alga Desmarestia accumulate high intracellular concentrations of acid (5,11,14,22). This acid accumulation is unusual in two respects; the pH (0.4-0.8) is considerably lower than that found in any other plant cell, and the acid is H2SO4 rather than an organic acid such as malic acid (14) Ion Concentration Determination. Tissue segments were rinsed and suspended for 10 min in either 0.5 M KC1 (for the Na+ and S042 assays) or 0.28 M Na2SO4 (for the K+ and C1-assays) at 0°C. Fresh weight was then determined and the segments frozen. Ions were extracted from the frozen segments via the boiling water method of Higinbotham et al. (7). The efficiency of this extraction method was checked by digesting the insoluble material in hot, concentrated HNO3 and assaying this for K+ and Na+. The average amount of either of these two ions remaining in the insoluble fraction was less than 0.1% of their total.Ion concentrations were determined as follows: K+ and Na+ by flame emission spectrophotometry, Cl-via potentiometric titration, and SO22 by precipitation with barium (added as BaCl2). Internal ion concentrations were estimated using, as the aqueous tissue volume, the fresh weight minus the dry weight on a 1 ml/g basis.Membrane Potential Measurement. Membrane potentials were measured using standard glass microelectrode techniques. The 3M KCl microcapillary salt bridges were connected through silversilver chloride wire to a Keithley Model 610B electrometer (input resistance 1014 ohms). Tissue segments were mounted in a tissue chamber with a continuous flow-through system for the temperature-controlled (10-12°C) bathing solution (artificial seawater). Because of the small cell size (10-20 ,um

Plants performing crassulacean acid metabolism show a large nocturnal accumulation of malic acid in the vacuole of the photosynthetic cells. It has been postulated that an H +-translocating ATPase energizes the transport of malic acid across the tonoplast into the vacuole. In the present work we have characterized the ATPase activity associated with vacuoles of the crassulacean-acid-metabolism plant Kalanchoe duigren~ontiurza and compare it with other phosphohydrolases.Vacuoles were isolated by polybase-induced lysis of mesophyll-ccll protoplasts. The vacuoles had a high activity of unspecific acid phosphatase (pH optimum 5.3). The acid phosphatase was strongly inhibited by ammonium molybdate (with 50 7; inhibition at about 0.5 mmol m-3), but was not completely inhibited even at much higher ammonium-molybdate concentrations. In contrast, the vacuolar ATPase activity, assayed in the presence of 100 inmol m S 3 ammonium molybdate, had a pH optimum of 8.0. ATP was the preferred substrate, but GTP, ITP and ADP were hydrolyzed a t appreciable rates. The mean ATPase activity at pH 8.0 was 14.5 nmol h-' (lo3 vacuoles)-', an average 13 : d of which was attributable to residual acid-phosphatase activity. Inorganicpyrophosphatase activity could not be demonstrated unambiguously.The vacuolar ATPase activity was M,$+-dependent, had an apparent K,,, for MgATP2-of 0.31 mol m-3, and was 32 YC stimulated by SO mol m-3 KCI. Ofthe inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO; were both markedly inhibitory. Dicyclohexylcarbodiiniide and tributyltin were also strongly inhibitory. Tributyltin caused a 50 '>A inhibition at about 0.3 mmol m-3. This is taken as evidcnce that the vacuolar ATPase might function as an H+-translocating ATPase. I t is shown that the measured activity of the vacuolar ATPase would be of the right order to account for the observed rates of nocturnal malic-acid accumulation in K. duigremontiuna.Many succulent plants exhibit a form of photosynthetic carbon assimilation known as crassulacean acid metabolism (CAM). These plants fix atmospheric CO, at night into malic acid. During the following day this malic acid is decarboxylated, and the released C 0 2 is assimilated via the photosynthetic carbon reduction cycle [I, 21. Following its synthesis at night in the cytoplasm, malic acid is transported across the tonoplast into the large, central vacuole of the photosynthetic cells [3]. This process is energy-requiring [3,4], whereas efflux of malic acid out of the vacuole is passive [5].The nature of the active-transport system responsible for malic-acid accumulation in the vacuole has not yet been elucidated. One possibility is that thc process is driven by a tonoplast ATPase that pumps protons from the cytoplasm into the vacuole, with the malate2-anions following passively to maintain electroneutrality [3.4]. This mechanism could satisfy the requirements for control ofcytoplasmic pH in the face of vacuolar pH values as low as 3.3 [6]. It would also be fe...