Effects of growth regulators on the induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum L.

The classical induction of Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum L. by water stress is observed within one week when fourto five-week-old plants (grown under a 16/8 h photoperiod at ca. 600 mol quanta · m^–2 · s^–1) are irrigated with 350 mM NaCl. The induction of CAM was evaluated by measuring phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) and NADP-malic enzyme (NADP-ME, EC 4.1.1.82) activities and nocturnal increases in malate content and titratable acidity of leaf extracts, and the daily pattern of CO₂ exchange and stomatal conductance during the 7-d induction period. Three growth regulators, abscisic acid (ABA), farnesol (an antitranspirant and analog of ABA), and benzylaminopurine (BAP), were found to substitute for NaCl for induction of CAM when fed to plants in nutrient media. Daily irrigation with solutions containing micromolar levels (optimum ca. 10 micromolar) of these growth regulators led to the induction of CAM similar to that by high salt. Application of the growth regulators, like NaCl, caused large increases in the activity of NADP-ME and the activity and level of PEPCase, which are components of the biochemical machinery required for CAM. Western immunoblotting showed that the increased activity of PEPCase on addition of ABA, farnesol and BAP was mainly due to increased levels of the CAM-specific isoforms. Also, dehydration of cut leaves over 8.5 h under light resulted in a severalfold increase in PEPCase activity. An equivalent increase in PEPCase activity in excised leaves was also obtained by feeding 150 mM NaCl, or micromolar levels of ABA or BAP via the petiole, which supports results obtained by feeding the growth regulators to roots. However, the increase in PEPCase activity was inhibited by feeding high levels of BAP to cut leaves prior to dehydration, indicating a more complex response to the cytokinin. Abscisic acid may have a role in induction of CAM in M. crystallinum under natural conditions as there is previous evidence that induction by NaCl causes an increase in the content of ABA, but not cytokinins, in leaves of this species.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CAM Crassulacean acid metabolism - Chl chlorophyll - 2,4D 2,4-dichlorophenoxyacetic acid - NADP-ME NADP-malic enzyme - PEPCase phosphoenolpyruvate carboxylase Methyl jasmonate was generously provided by Dr. Vincent Franceschi (Botany Department, Washington State University). The anti-maize leaf PEPCase was kindly supplied by Dr. Tatsuo Sugiyama (Department of Agricultural Chemistry, Nagoya University, Japan) and the anti-Flaveria trinervia leaf PEPCase was kindly supplied by Dr. Samuel Sun (Department of Plant Molecular Physiology, University of Hawaii, Honulu). This work was funded in part by U.S. Department of Agriculture Competitive Grant 90-37280-5706 and an equipment grant (DMB 8515521) from the National Science Foundation. Ziyu Dai was supported in part by Guangxi Agricultural College and Ministry of Agriculture of the People's Republic of China     

Properties of phosphoenolpyruvate carboxylase in rapidly prepared,desalted leaf extracts of the Crassulacean acid metabolism plant Mesembryanthemum crystallinum L.

Properties of phosphoenolpyruvate (PEP) carboxylase, obtained from leaves of Mesembryanthemum crystallinum L. performing Crassulacean acid metabolism (CAM), were determined at frequent time points during a 12-h light/12-h dark cycle. Leaf extracts were rapidly desalted and PEP carboxylase activity as a function of PEP concentration, malate concentration, and pH was measured within 2 min after homogenization of the tissue. Maximum velocity of PEP carboxylase was similar in the light and dark at pH 7.5 and pH 8.0. However, PEP carboxylase had as much as a 12-fold lower K _m for PEP and as much as a 20-fold higher K _i for malate during the dark than during the light periods, the magnitude of these differences being dependent on the assay pH. Assuming that enzyme properties immediately after isolation reflect the approximate state of the enzyme in vivo, these differences in enzyme properties reduce the potential for CO₂ fixation via PEP carboxylase in the light. A small decrease in cytoplasmic pH in the light would greatly magnify the above differences in day/night properties of PEP carboxylase, because the sensitivity of PEP carboxylase to inhibition by malate increased with decreasing pH. Properties of PEP carboxylase were also studied in plants exposed to short-term perturbations of the normal 12-h light/12-h dark cycle (e.g., prolonged light period, prolonged dark period). Under all light/dark regimes, there was a close correlation between change in properties of PEP carboxylase and changes of the tissue from acidification to deacidification, and vice versa. Changes in properties of PEP carboxylase were not merely light/dark phenomena because they were also observed in plants exposed to continuous light or dark. the data indicate that, during CAM, PEP carboxylase exists in two stages which differ in their capacity for net malate synthesis. The physiologically-active state is distinguished by a low K _m for PEP and a high K _i for malate and favors malate synthesis. The physiologically-inactive state has a high K _m for PEP and a low K _i for malate and exists during periods of deacidification and other periods lacking synthesis of malic acid.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPC PEP carboxylase - RuBP ribulose 1,5-bisphosphate - RH relative humidity     

Na+/H+-transporter, H+-pumps and an aquaporin in light and heavy tonoplast membranes from organic acid and NaCl accumulating vacuoles of the annual facultative CAM plant and halophyte Mesembryanthemum crystallinum L.

Crassulacean acid metabolism (CAM) was induced in Mesembryanthemum crystallinum L. by either NaCl- or high light (HL)- stress. This generated in mesophyll cells predominantly of NaCl-stressed plants two different types of vacuoles: the generic acidic vacuoles for malic acid accumulation and additionally less acidic (“neutral”) vacuoles for NaCl sequestration. To examine differences in the tonoplast properties of the two types of vacuoles, we separated microsomal membranes of HL- and NaCl-stressed M. crystallinum plants by centrifugation in sucrose density gradients. Positive immunoreactions of a set of antibodies directed against tonoplast specific proteins and tonoplast specific ATP- and PP_i-hydrolytic activity were used as markers for vacuolar membranes. With these criteria tonoplast membranes were detected in both HL- and NaCl-stressed plants in association with the characteristic low sucrose density but also at an unusual high sucrose density. In HL-stressed plants most of the ATP- and PP_i-hydrolytic activity and cross reactivity with antibodies including that directed against the Na⁺/H⁺-antiporter from Arabidopsis thaliana was detected with light sucrose density. This relationship was inverted in NaCl-stressed plants; they exhibited most pump activity and immunoreactivity in the heavy fraction. The relative abundance of the heavy membrane fraction reflects the relative occurrence of “neutral” vacuoles in either HL- or NaCl-stressed plants. This suggests that tonoplasts of the “neutral” vacuoles sediment at high sucrose densities. This is consistent with the view that this type of vacuoles serves for Na⁺ sequestration and is accordingly equipped with a high capacity of proton pumping and Na⁺ uptake via the Na⁺/H⁺-antiporter.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Phenotypic changes in the fluidity of the tonoplast membrane of crassulacean-acid-metabolism plants in response to temperature and salinity stress

Electron paramagnetic resonance-spectroscopic studies on spin-labeled purified tonoplast membranes showed that in the obligate crassulacean-acid-metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perr. the fluidity of the tonoplast decreased during acclimation to higher temperatures. This phenotypic change in tonoplast fluidity was paralleled by a decrease in the mobilization of malic acid from the vacuoles during CAM in the light. The shift from the C₃ to the CAM mode of photosynthesis in the facultative CAM plant Mesembryanthemum crystallinum L. also led to a decrease in the fluidity of the tonoplast membrane. The results are consistent with the hypothesis that the ability to store malic acid during CAM in the vacuoles depends largely on the actual fluidity of the tonoplast membrane.     

A plant lipid and the platelet-activating factor stimulate ATP-dependent H+ transport in isolated plant membrane vesicles

A plant lipid was isolated from zucchini (Cucurbita pepo L.) membranes and from soybean (Glycine max [L.] Merr) phospholipids by thinlayer chromatography and further purified by high-performance liquid chromatography. This plant lipid was chromatographically very similar to the platelet-activating factor, an ether phospho-lipid with hormone-like properties found in mammals. Both the plant lipid and the platelet-activating factor stimulated ATP-dependent H⁺ transport in isolated membrane vesicles from zucchini hypocotyls.Abbreviations HPLC high-performance liquid chromatography - PAF platelet-activating factor     

Separation and purification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible Crassulacean acid metabolism

Tonoplast vesicles were isolated from Kalanchoe daigremontiana Hamet et Pierrer de la Bâthie and Mesembryanthemum crystallinum L., exhibiting constitutive and inducible crassulacean acid metabolism (CAM), respectively. Membrane-bound proteins were detergent-solubilized with 2% of Triton X-100. During CAM induction in M. crystallinum, ATPase activity increases four-fold, whereas pyrophosphatase activity decreases somewhat. With all plants, ATPase and pyrophosphatase could be separated by size-exclusion chromatography (SEC, Sephacryl S 400), and the ATPase was further purified by diethylaminoethyl-ion-exchange chromatography. Sodium-dodecyl-sulfate electrophoresis of the SEC fractions from K. daigremontiana containing maximum ATPase activity separates several protein bands, indicating subunits of 72, 56, 48, 42, 28, and 16 kDa. Purified ATPase from M. crystallinum in the C₃ and CAM states shows a somewhat different protein pattern. With M. crystallinum, an increase in ATP-hydrolysis and changes in the subunit composition of the native enzyme indicate that the change from the C₃ to the CAM state is accompanied by de-novo synthesis and by structural changes of the tonoplast ATPase.Abbreviations CAM Crassulacean acid metabolism - DTT dithiothreitol - kDa kilodalton - PAGE polyacrylamide gel electrophoresis - PPiase pyrophosphatase - SEC size exclusion chromatography - SDS sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Essential sulfhydryl groups in the catalytic center of the tonoplast H+-ATPase from coleoptiles ofZea mays L. as demonstrated by the biotin-streptavidin-peroxidase system

Functional properties and the localization of essential SH-groups of the tonoplast H⁺-ATPase fromZea mays L. were studied. In contrast to the pyrophosphate-dependent H⁺-translocation activity of the tonoplast, the H⁺-ATPase activity was inhibited by SH-blocking agents, such as N-ethylmaleimide and iodoacetic acid. In the case ofp-hydroxymercuribenzoate, HgCl₂ and oxidized glutathione, the inhibition could be reversed by adding reduced glutathione or dithiothreitol. Incubation of tonoplast vesicles with oxidized glutathione or N-ethylmaleimide in the presence of Mg·ADP—a competitive inhibitor of the ATP-dependent H⁺ pump—avoided the inhibition of the H⁺-pumping activity. This effect is an indication for the occurrence of essential SH-groups at the catalytic site of the H⁺-ATPase. In order to characterize the active center these thiols were specifically labeled with maleimidobutyrylbiocytin. Subsequently, the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an immobilizing membrane. The maleimidobutyrylbiocytin-labeled active-center protein was detected by a biotin-streptavidin-peroxidase staining system and was shown to be a 70-kDa subunit of the tonoplast H⁺-ATPase. It is suggested that the oxidation state of the critical sulfhydryl groups within the active center of the enzyme and their reversible blocking by endogenous compounds might be of great importance for the regulation of the enzyme activity in vivo.     

Nuclear-magnetic-resonance imaging of leaves ofMesembryanthemum crystallinum L. plants grown at high salinity

Differences in water binding were measured in the leaf cells ofMesembryanthemum crystallinum L. plants grown under high-salinity conditions by using nuclear-magnetic-resonance (NMR) imaging. The 7-Tesla proton NMR imaging system yielded a spatial resolution of 20·20·100 m³. Images recorded with different spin-echo times (4.4 ms to 18 ms) showed that the water concentrations in the bladder cells (located on the upper and lower leaf surface), in the mesophyll cells and in the water-conducting vessels were nearly identical. All of the water in the bladder cells and in the water-conducting vessels was found to be mobile, whilst part of the water in the mesophyll cells was bound. Patches of mesophyll cells could be identified which bound water more strongly than the surrounding mesophyll cells. Optical investigations of leaf cross-sections revealed two types of mesophyll cells of different sizes and chloroplast contents. It is therefore likely that in the small-sized mesophyll cells water is strongly bound. A long-term asymmetric water exchange between the mesophyll cells and the bladder cells during Crassulacean acid metabolism has been described in the literature. The high density of these mesophyll cells in the lower epidermis is a possible cause of this asymmetry.Abbreviations CAM Crassulacean acid metabolism - NMR nuclear magnetic resonance - T_E spin-echo time     

An ATP-driven proton pump in brush-border membranes from rat renal cortex

Summary The rate of ATP hydrolysis in ATP-preloaded plasma membrane vesicles derived from the luminal membrane of renal cortical tubules, and the rate of H⁺ secretion out of the same vesicles were investigated. Both were inhibited at low temperature, by the action of filipin, an antibiotic that complexes with cholesterol in plasma membranes, and by the action of blockers of mitochondrial F_o hydrogen channels, dicyclohexylcarbodiimide and Dio-9. Valinomycin in the presence of K⁺ showed a stimulatory effect, the protonophor carbonyl-cyanid-p-trifluormethoxy-phenylhydrazone stimulated the intravesicular ATP hydrolysis and apparently abolished acidification of the extravesccular medium. Lowering of the pH of the extravesicular medium retarded ATP hydrolysis, while readjustment of extra- and intravesicular pH accelerated ATP hydrolysis again. These findings strongly support the assumption that an ATP-driven proton pump is located in the luminal membrane of renal cortical tubules.     

Active transport of Ca in membrane vesicles from pea: Evidence for a H/Ca antiport

Two non mitochondrial systems involved in ATP-dependent Ca²⁺ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca²⁺ accumulation system, which shows the characteristics of an ATP-dependent H⁺/Ca²⁺ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO₄-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg²⁺ or Mn²⁺. A second system of ATP-dependent Ca²⁺ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO₄-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg²⁺ or Mn²⁺. This system is interpreted as corresponding to the (Ca²⁺ + Mg²⁺)-ATPase described by Dieter, P. and Marmé, D. ((1980) Planta 150, 1–8).     

Some characteristics of anion transport at the tonoplast of oat roots,determined from the effects of anions on pyrophosphatedependent proton transport

The effects of anions on inorganicpyrophosphate-dependent H⁺-transport in isolated tonoplast vesicles from oat (Avena sativa L.) roots were determined. Both fluorescent and radioactive probes were used to measure formation of pH gradients and membrane potential in the vesicles. Pyrophosphate hydrolysis by the H⁺-translocating pyrophosphatase was unaffected by anions. Nonetheless, some anions (Cl^-, Br^- and NO₃-) stimulated H⁺-transport while others (malate, and iminodiacetate) did not. These differential effects were abolished when the membrane potential was clamped at zero mV using potassium and valinomycin. Stimulation of H⁺-transport by Cl^- showed saturation kinetics whereas that by NO₃- consisted of both a saturable component and a linear phase. For Cl^- and NO₃-, the saturable phase had a K _m of about 2 mol·m^-3. The anions that stimulated H⁺-transport also dissipated the membrane potential (.) generated by the pyrophosphatase. It is suggested that the stimulatory anions cross the tonoplast in response to the positive generated by the pyrophosphatase, causing dissipation of and stimulation of pH, as expected by the chemiosmotic hypothesis. The work is discussed in relation to recent studies of the effects of anions on ATP-dependent H⁺-transport at the tonoplast, and its relevance to anion accumulation in the vacuole in vivo is considered.Abbreviations and symools BTP 1,3-bis[tris(hydroxymethyl)-methylamino]-propane - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - IDA iminodiacetate - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi morganic pyrophosphate     

The use of a chloride-sensitive fluorescent probe to measure chloride transport in isolated tonoplast vesicles

A fluorescence method for the direct measurement of Cl^- transport in isolated tonoplast vesicles is described. This technique utilises the Cl^--sensitive fluorescent compound, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). This is a water-soluble compound with excitation and emission wavelengths of 350 and 440 nm, respectively. Its fluorescence is quenched by Cl^-, Br^-, I^-, SCN^-, NO ₂ ^- and tetraphenylborate but not by NO ₃ ^- , SO ₄ ^2- , iminodiacetate or malate. These effects are independent of pH. This compound was loaded into tonoplast vesicles from red beet (Beta vulgaris L.) storage roots or from barley (Hordeum vulgare L.) roots by incubation at 37° C and the external probe was then removed by repeated centrifugation of the vesicles in SPQ-free medium. In this way a large proportion of the observed fluorescence signal was from the interior of the vesicles, and its quenching could be used to monitor, quantitatively, and in real time, the intravesicular Cl^- concentration. In this paper we describe some of the problems encountered in using this probe to measure Cl^- transport in tonoplast vesicles, how these were overcome and some characteristics of Cl^- transport at the tonoplast as measured by the probe.Abbreviations and symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino-propane - DTT dithiothreitol - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi inorganic pyrophosphate - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Proton and anion transport at the tonoplast in crassulacean-acid-metabolism plants: specificity of the malate-influx system in Kalanchoë daigremontiana

Tonoplast vesicles were prepared from leaf mesophyll homogenates of the crassulacean-acid-metabolism plant Kalanchoë daigremontiana Hamet et Perrier de la Bâthie to study the effects of anions on ATP- and inorganic-pyrophosphate (PPi)-dependent H⁺ transport. In the presence of gramicidin, substrate hydrolysis by the tonoplast ATPase was characteristically stimulated by chloride and inhibited by nitrate, but was unaffected by malate and a wide range of other organic-acid anions; the PPiase was anion-insensitive. Malate was more effective than chloride both in stimulating ATP- and PPi-dependent vesicle acidification (measured as quinacrine-fluorescence quenching) and in dissipating a pre-existing inside-positive membrane potential (measured as oxonol-V-fluorescence quenching), indicating that malate was more readily transported across the tonoplast. Certain other four-carbon dicarboxylates also supported high rates of vesicle acidification, their order of effectiveness being fumarate malate -succinate > oxalacetate - tartrate; the five-carbon dicarboxylates 2-oxoglutarate and glutarate were also transported, although at lower rates. Experiments with non-naturally occurring anions indicated that the malate transporter was not stereospecific, but that it required the trans-carboxyl configuration for transport. Shorter-chain or longer-chain dicarboxylates were not transported, and neither were monocarboxylates, the amino-acid anions aspartate and glutamate, nor the tricarboxylate isocitrate. The non-permeant anions maleate and tartronate appeared to be competitive inhibitors of malate transport but did not affect chloride transport, indicating that malate and chloride influx at the tonoplast might be mediated by separate transporters.Abbreviations BTP 1,3-bis-[tris(hydroxymethyl)methylamino]-propane - CAM crassulacean acid metabolism - oxonol V bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol - pH transmembrane pH difference - PPi inorganic pyrophosphate - PPiase inorganic pyrophosphatase - quinacrine 6-chloro-9-{[4-(diethylamino)-1-methylbutyl]amino}-2-methoxyacridine dihydrochloride - transmembrane electrical potential difference     

The mechanism of nitrate transport across the tonoplast of barley root cells

Nitrate-selective microelectrodes were used to measure not only nitrate activity in the cytoplasm and vacuole of barley (Hordeum vulgare L.) root cells, but also the tonoplast electrical membrane potential. For epidermal cells, the mean cytoplasmic and vacuolar pNO₃ (-log₁₀ [NO₃]) values were 2.3±0.04 (n=19) and 1.41±0.03 (n=35), respectively, while for cortical cells, the mean cytoplasmic and vacuolar nitrate values were 2.58±0.18 (n=4) and 1.17±0.06 (n=13), respectively. These results indicate that the accumulation of nitrate in the vacuole must be an active process. Proton-selective microelectrodes were used to measure the proton gradient across the tonoplast to assess the possibility that nitrate transport into the vacuole is mediated by an H⁺/NO ₃ ^– antiport mechanism. For epidermal cells, the mean cytoplasmic and vacuolar pH values were 7.12±0.06 (n=10) and 4.93±0.11 (n=22), respectively, while for cortical cells, the mean cytoplasmic and vacuolar pH values were 7.24±0.07 (n=3) and 5.09±0.17 (n=7), respectively. Calculations of the energetics for this mechanism indicate that the observed gradient of nitrate across the tonoplast of both epidermal and cortical cells could be achieved by an H⁺/NO ₃ ^– antiport with a 11 stoichiometry.Abbreviations and Symbols G/F free-energy change for H⁺/NO ₃ ^– antiport - F Faraday constant - pH_c cytoplasmic pH - pH_v vacuolar pH - p[NO₃]_c log₁₀ (cytoplasmic [NO ₃ ^– ]) - P[NO₃]_v -log₁₀ (vacuolar [NO₃]) We wish to thank Dr. K. Moore for assistance with statistical analysis.     

The function of tonoplast ATPase in intact vacuoles of red beet is governed by direct and indirect ion effects

The pH gradient and the electric potential across the tonoplast in mechanically isolated beetroot vacuoles has been studied by following the uptake of [¹⁴C]methylamine and [¹⁴C]triphenyl-methylphosphoniumchloride. In response to Mg-ATP, the vacuolar interior is acidified by 0.8 units. This strong acidification is accompanied by a slight hyperpolarization of the membrane potential, which is probably caused by a proton diffusion potential. In preparations where only a small acidification (0.4 units) occurred, the membrane potential was depolarized by the addition of Mg-ATP. Different monovalent cations and anions were tested concerning their effect on the pH gradient and ATPase activity in proton-conducting tonoplasts. Chloride stimulation and NO ₃ ^- inhibition were clearly present. The observed decline of the pH gradient upon the addition of Na⁺ salts is probably caused by an Na⁺/H⁺ antiport system.Abbreviations and symbol CCCP carbonylcyanide-m-chlorophenylhydrazone - Mes 2(N-morpholino)ethanesulfonic acid - TPMP⁺ triphenylmethylphosphoniumchloride - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - membrane potential Dedicated to Professor A. Betz on the occasion of his 65th birthday     

Photosynthesis in enzymatically isolated leaf cells from the CAM plant Sedum telephium L.

A technique has been developed for the enzymatic isolation of leaf cells from the Crassulacean acid-metabolism plant Sedum telephium. The cells exhibited high activity in both ¹⁴CO₂ incorporation (30–70 mol CO₂ mg^-1 chlorophyll h^-1) and O₂ evolution in the presence of bicarbonate (60–110 mol O₂ mg^-1 chlorophyll h^-1). Half-maximum saturation of ¹⁴CO₂ incorporation occurred at a bicarbonate concentration of ca. 2 mM (20 M CO₂) at pH 8.4 and 30°C. Two types of light-dependent O₂ evolution are reported: O₂ evolution in the absence of exogenously supplied bicarbonate (endogenous O₂ evolution), and bicarbonate-stimulated O₂ evolution. Oxygen evolution in the presence of approximately ambient concentrations of CO₂ appeared to be a combination of the endogenous O₂ evolution and O₂ evolution from fixation of the exogenously supplied CO₂.Abbreviations CAM Crassulacean acid metabolism - cirlo chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - RuDP ribulose-1,5-diphosphate     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.