The Regulation of Citric Acid Accumulation and Carbon Recycling During CAM in Ananas comosus

The carbon balance of shade-grown Ananas comosus was investigatedwith regard to nitrogen supply and responses to high PAR. Netdark CO₂ uptake was reduced from 61.2 to 38.5 mmol CO₂ m^–2in N limited (–N) plants grown under low PAR (60 µmolm^–2 s^–1) and apparent photon yield declined from0.066 to 0.034 (mol 0².mol^–1 photon), although photosyntheticcapacities (measured under 5% CO₂) were similar. Following transferfor 7 d to high PAR (600. µmol m^–2 s^–1), netCO² uptake at night increased by 14% in +N plants, and daytimephotosynthetic capacity was higher, with a maximum value of7.8 µmol m^–2 s^–1. The magnitude of dark CO₂ fixation during CAM was measured asdawn—dusk variations in leaf-sap titratable acidity (H+)and as the proportion of malic and citric acids. The contributionfrom re-fixation of respiratory CO₂ recycling (measured as thedifference between net CO₂ uptake and malic acid accumulation)varied with growth conditions, although it was generally lower(30%) than reported for other bromeliads. Assuming a stoichiometryof 2H+: malate and 3H+: citrate, there was a good agreementbetween titratable protons and enzymatically determined organicacids. The accumulation of citric acid was related to nitrogensupply and PAR regime, increasing from 7.0 mol m–3 (+Nplants) to 18 mol m^–3 (–N plants) when plants weretransferred to high PAR; malate: citrate ratios decreased from13.1 to 2.5 under these conditions. Under the low PAR regime, leaf-sap osmotic pressure increasedat night in proportion to malic acid accumulation. However,following the transfer to high PAR for 7 d, there was a muchgreater depletion of soluble sugars at night which correspondedto a decrease in leaf-sap osmotic pressure. Although a rolefor citric acid in CAM has not been properly defined, it appearsthat the accepted stoichiometry for CAM in terms of gas exchange,titratable acidity, malic acid and osmotic pressure may nothold for plants which accumulate citric acid. Key words: Ananas comosus, CAM, citric acid accumulation, carbon recycling     

Effects of Cl- on and Malate Fluxes and CO2 Fixation in Carrot and Barley Root Cells

Cl^– and ions interact apparently competitively during influx across the plasmalemma of carrot root cells. Cl^–,however, reduces influx much less than predicted from the effect of on Cl^– influx.Cl^– and plasmalemma influxes both increase with time after excision of carrot tissue. Cl^–and may therefore be transported by a common mechanism. The effect of pH changes on the influx of malate across theplasmalemma in barley roots shows that malate crosses the plasmalemmaas the singly charged anion. Stimulations of influx by bothK₂SO₄ and KCl suggest that the malate anion crosses in associationwith. K⁺. If malate entry is passive, P_mal- is about 2?10^–8cm s^–1, but it is thought that malate entry is partlyan active process. A slight, apparently competitive inhibition by Cl^– ofmalate flux into the vacuole of barley root cells suggests thatthe two anions may be transported by a common process at thetonoplast, but this is not thought to be physiologically significant. The accumulation of ¹⁴C from 1 mM is drastically reduced by 10 mM Cl^–. A quantitative analysis of the kineticsof ¹⁴C exchange shows that Cl^– directly inhibits the formationof malate from . The decreased influx of endogenously produced malate to the vacuole in the presenceof Cl^– is probably a secondary consequence of the fallin the cytoplasmic concentration. The nature of the Cl^–inhibition of malate formation is discussed. In KCl-loaded tissue the influx of external malate and the accumulationof ¹⁴C from external are reduced. The location of these effects is not certain, but the effects suggest thatregulation of malate synthesis and accumulation may be relatedto the negative-feedback regulation of Cl^– and transport.     

Properties of Phosphoenolpyruvate Carboxylase and Carbohydrate Accumulation in the C3-CAM Intermediate Sedum telephium L. Grown Under Different Light and Watering Regimes

A comparison of the activity and properties of the enzyme phosphoenolpyruvatecarboxylase (PEPC) was made for plants of Sedum telephium L.grown under low (70 µmol m^–2 s^–1) or high(500µmol m^–2 s^–1) PPFD and subjected to varyingdegrees of water stress. Under well-watered conditions onlyplants grown under high PPFD accumulated titratable acidityovernight and the extractable activity of PEPC was almost 2-foldhigher in these plants than in plants grown under low PPFD.Increasing drought stress resulted in a substantial increasein the activity of PEPC extracted both during the light anddark periods and a decrease in the sensitivity to inhibitionby malic acid. The magnitude of these changes was determinedby the severity and duration of drought and by light intensity.A comparison of the kinetic properties of PEPC from severelydroughted plants revealed that plants droughted under high PPFDhad a lower K_m for PEP than plants under low PPFD. Additionof 2·0 mol m^–3 malate resulted in an increase inthe K_m for PEP, with plants draughted under low PPFD havinga significantly higher K_m in the presence of malic acid comparedto those under high _PPFD. Response to the activator glc-6-P,which lowered the K_m for PEP, also varied between plants grownunder the two light regimes. Under well-watered conditions PEPCextracted from plants under high PPFD was more sensitive toactivation by glc-6-P than those under low PPFD. After the severedrought treatment, however, the K_m for PEP in the presence ofglc-6-P was similar for enzyme extracted from plants grown underboth light regimes. Soluble sugars and starch were depletedovernight and were both possible sources of substrate for PEPC.With increasing drought, however, the depletion of starch relativeto soluble sugars increased under both light regimes. The propertiesof PEPC and the characteristics of carbohydrate accumulation/depletionare discussed in relation to the regulation of CAM in S. telephiumgrown under different light and watering regimes. Key words: PEP carboxylase, CAM, carbohydrates, Sedum telephium     

Salinity-induced Malate Accumulation in Chara

Ion absorption by Chara corallina from solutions containingpredominantly KC1 or RbCl at up to 100 mol m^–3 resultedin accumulation of salts and turgor regulation. Turgor regulationdid not occur in solutions containing Na⁺ or Li⁺salts. Duringion absorption from various salts of K⁺ and Rb⁺ vacuolar cationconcentration exceeded Cl^– concentration. This differencewas shown to be balanced by the synthesis and accumulation ofmalate. Vacuolar malate concentration reached 48 mol m₃^–,with accumulation occurring at rates of up to 0.45 mol m^–3h^–1. Malate accumulation was inhibited by low externalpH and was dependent upon external HCO₃^– concentration.The synthesis of malic acid and its subsequent dissociationimposed a severe acid load on the cell. Biophysical regulationof cellular pH was achieved by a H⁺efflux at a rate of about40 nmol m^–2 s^–1from the cell. The results presentedargue against cytoplasmic Cl^–, HCO₃^– or pH regulatingmalate accumulation in Chara and it is suggested that malatetransport across the tonoplast may regulate malate accumulation. Key words: Malate, Chara corallina, pH regulation, salinity     

Stimulatory Effect of Chloride Ions on NADP Malic Enzyme from Leaves of two C4 Species of Cyperus

NADP malic enzyme (EC 1.1.1.40 [EC] ) from leaves of two C₄ speciesof Cyperus (C. rotundus and C. brevifolius var leiolepis) exihibiteda low level of activity in an assay mixture that contained lowconcentrations of Cl^–. This low level of activity wasmarkedly enhanced by increases in the concentration of NaClup to 200 mM. Since the activity of NADP malic enzyme was inhibitedby Na₂SO₄ and stimulated by relatively high concentration ofTris-HCl (50–100 mM, pH 7–8), the activation ofthe enzyme by NaCl appears to be due to Cl^–. Variationsin the concentration of Mg²⁺ affected the K_A (the concentrationof activator giving half-maximal activation) for Cl^–,which decreased from 500 mM to 80 mM with increasing concentrationsof Mg²⁺ from 0.5 mM to 7 mM. The K_m for Mg²⁺ was decreased from7.7 mM to 1.3 mM with increases in the concentration of NaClfrom zero to 200 mM, although the increase of V_max was not remarkable.NADP malic enzyme from Cyperus, being similar to that from otherC₄ species, was able to utilize Mn²⁺. The K_m for Mn²⁺ was 5mM, a value similar to that for Mg²⁺. The addition of 91 mMNaCl markedly decreased the K_m for Mn²⁺ to 20 +M. NADP malicenzyme from Setaria glauca, which contains rather less Cl^–than other C₄ species, was inactivated by concentrations ofNaCl above 20 mM, although slight activation of the enzyme wasobserved at low concentrations of NaCl at pH7.6. (Received February 20, 1989; Accepted June 12, 1989)     

The Effect of Temperature and Light on Gas Exchange and Acid Accumulation in the C3-CAM Plant Clusia minor L

Gas exchange and organic acid accumulation of the C₃-CAM intermediateClusia minor L. were investigated in response to various day/nighttemperatures and two light regimes (low and high PAR). For bothlight levels equal day/night temperatures between 20°C and30°C caused a typical C₃ gas exchange pattern with all CO₂uptake occurring during daylight hours. A day/ night temperatureof 15°C caused a negative CO₂ balance over a 24 h periodfor low-PAR-grown plants while high-PAR-grown plants showeda CAM gas exchange pattern with most CO₂ uptake taking placeduring the dark period. However, there was always a considerablenight-time accumulation of malic acid which increased when thenight-time temperature was lowered and had its maximum (54 mmolm^–2) at day/night temperature of 30/15°C. A significantamount of malic acid accumulation (23 mmol m^–2) in low-PAR-grownplants was observed only at 30/15°C. Recycling of respiratoryCO₂ in terms of malic acid accumulation reached between 2·0and 21·5 mmol m_–2 for high-PAR-grown plants whilethere was no significant recycling for low-PAR-grown plants.Both low and high-PAR-grown plants showed considerable night-timeaccumulation of citric acid. Indeed under several temperatureregimes low-PAR-grown plants showed day/night changes in citricacid levels whereas malic acid levels remained approximatelyconstant or slightly decreased. It is hypothesized that lowand high-PAR-grown plants have different requirements for citrate.In high-PAR-grown plants, the breakdown of citrate preventsphotoinhibition by increasing internal CO₂ levels, whereas inlow-PAR-grown plants the night-time accumulation of citric acidmay function as an energy and carbon saving mechanism. Key words: C. minor, C₃, CAM, citric acid, light intensity     

Free Ca2+ in tissue 22+ -selective electrodes

Calcium was measured with Ca²⁺ -selective electrodes in calciotrophicCAM plants. Several organic acid anions (citrate, isocitrate,malate, and malonate) were tested for their capacity to chelateCa²⁺ in solutions at pH 4.8. Free Ca²⁺ was also calculated fromthe stability constants of the chelates at pH 4.0 and pH 6.0.The strongest chelator at pH 4.8 was citrate, reducing freeCa²⁺ from 10 to 0.5 mol m^–3, while isocitrate and malatedecreased ionized Ca²⁺ to 25% and 5O%, respectively. At pH 4.0isocitrate is somewhat more effective than citrate. Malonatehas only slight effects on free Ca²⁺ at pH 6.0. In tissue sapsfrom field-grown species of Crassula sp., Crassula expansa,Aloe ramosissima, and Aloe pillansii, concentrations of totalwater-soluble Ca²⁺ ranged from 25 to 196 mol m^–3. Measurementswith Ca²⁺ -selective electrodes showed that ionized Ca²⁺ wasreduced to 62–88% in the presence of isocitrate. Diurnalfluctuations in malate were less important for Ca²⁺ chelation,which was also true for the situation in greenhouse-grown plantsof Kalancho daigremontiana, which were cultivated at differentCa²⁺ levels. Comparing the osmotic potentials measured in thetissue saps with those calculated from the concentrations ofthe different solutes gave further evidence for the chelationof Ca²⁺ by organic acid anions, since values for using onlythe free Ca²⁺ were much closer to the measured values of thanthose calculated with total water-soluble Ca²⁺. Key words: Calciotrophic plants, CAM, organic acids, free Ca²⁺, Ca²⁺ chelation     

The Damping and Reinitiation of the Circadian Rhythm of CO2 Output in Bryophyllum Leaves in Relation to their Malate Content

The circadian rhythm of CO₂ output in leaves of Bryophyllumfedtschenkoi damps out after 3–4 d in continuous darknessand a CO₂-free air stream at 15°C. The rhythm is reinitiatedafter a single exposure to white light of 2, 4, 6 or 8 h duration,damps out again after a further 3–4 d and can be reinitiatedfor a second time by a further exposure to light. During the exposure to light there is a burst of CO₂ outputconsistent with the decarboxylation of malate, and the rhythmbegins afterwards with an initial high rate of CO₂ fixation.Malate gradually accumulates in the leaves in continuous darknessto attain a maximum value (35 mol m^–3) at the time whenthe circadian rhythm disappears, and decreases to a low value(19 mol m^–3) after a 4 h exposure to light which reinitiatesrhythmicity. These results support the hypothesis that damping of the rhythmof CO₂ output in continuous darkness is due to the accumulationof malate in the leaf cells, eventually reaching such a levelthat its removal from the cytoplasm into the vacuole cannottake place, with the result that PEPc activity, upon which therhythm of CO₂ output depends, remains allosterically inhibited. Key words: CAM, circadian rhythm, Bryophyllum, CO₂-fixation, malate metabolism     

Pinitol, a Compatible Solute in Mesembryanthemum crystallinum L.?

The irrigation of Mesembryanthemum crystallinum L. plants with400 mol m^–3 NaCl to induce crassulacean acid metabolism(CAM) was accompanied by the accumulation of pinitol. Pinitolconstituted 71% of the soluble carbohydrate fraction and 9.7%dry weight in the CAM form. Pinitol in the C₃ form did not exceed5% of the soluble carbohydrate fraction. Pinitol appeared metabolicallyinert: it was not readily degraded during 96 h of darkness inthe CAM form or during CAM deinduction. Preparations of CAMM. crystallinum protoplasts, vacuoles and chloroplasts showedpinitol to be chloroplastic at a concentration of about 230mol m^–3 and cytosolic at about 100 mol m^–3. No pinitolwas detected in vacuoles. CAM leaf extracts possessed a highermyo-inositol phosphate synthesising capacity than C₃ extracts,revealing greater activity in the CAM form of glucose-6-phosphatecycloaldolase, an enzyme in the pathway of pinitol synthesis. Although pinitol accumulation and CAM induction could not beseparated and appeared to be specific responses to water stress,there may not be a causal link between them. Pinitol may functionas a compatible solute in the cytosol and especially the chloroplaststo counteract the presence of high concentrations of Na⁺ andCl^– ions in the vacuole. The accumulation of pinitol,though apparently not directly related to CAM may, like CAM,be viewed as an aspect of the adaptation of the plant to a reductionin water availability. Key words: pinitol, Mesembryanthemum crystallinum L, CAM, compatible solute     

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

Induction and Repression of CAM in Sedum telephium L. in Response to Photoperiod and Water Stress

Lee, H. S. J. and Griffiths, H. 1987. Induction and repressionof CAM in Sedurn relephluni L. in response to photopcnod andwater stress.—J. exp. Bot. 38: 834–841. The introduction and repression of CAM in Sedurn telephiunmL, a temperate succulent, was investigated in watered, progressivelydrouglited and rewatered plants in growth chambers. Measurementswere made of water vapour and CO₂ exchange, titratable acidity(TA) and xylem sap tension. Effects of photoperiod were alsostudied. CAM was induced by drought under long or short days,although when watered no CAM activity was expressed. C₃-CAM intermediate plants were used for the investigation ofwater supply. Those which had received water and those drought-stressedboth displayed a similar nocturnal increase in TA, with a day-nightmaximum (H+) of 69 µmol g^–1 fr. wt. The wateredplants took up CO₂ at a maximum rate of 2?2 µmol m^–2s^–1 only in the light period, while the droughted plantsshowed a maximum nocturnal CO₂ uptake rate of 0?69 µmolm^–2 s^–1. Subsequently, as CAM was repressed, thewatered S. telephiwn displayed little variation in TA, withconstant levels at 42 µmol g^–1 fr. wt. (day 10).After 10 d of drought stress, the CAM characteristics of S.telephiurn were aLso affected, with reduced net CO₂ uptake andH+. The transition between C₃ and CAM in S. telephium can be describedas a progression in terms of the proportion of respiratory CO₂which is recycled and refixed at night as malic acid, in comparisonwith net CO₂ uptake. Recycling increased from 20% (day 1) to44% (day 10) as a result of the drought stress and was highin both the CAM-C₃ stage (no net CO2 uptake at night) and alsoin the drought-stressed CAM stage (reduced net CO₂ uptake atnight). The complete C₃-CAM transition occurred in less than8 d, and the stages could be characterized by xylem sap tensionmeasurements: CAM = 0?50 MPa C₃-CAM = 0?36 MPa C₃ = 0?29 MPa. Key words: CAM, Sedum telephium L., recycling     

Mechanism of passive malic-acid efflux from vacuoles of the CAM plantKalanchoë daigremontiana

Summary An analysis was carried out of the mechanism of malic-acid efflux from vacuoles of mesophyll cells of the crassulacean acid metabolism (CAM) plantKalanchoë daigremontiana. Following its accumulation in the vacuole as a result of nocturnal CO₂ fixation, the malic acid is passively transported back across the tonoplast in the subsequent light period and is decarboxylated in the cytoplasm. Malic-acid efflux was studied using leaf slices in solution or by following malic-acid utilization (deacidification) in leaves of intact plants. Samples of leaf-cell sap were taken at different times during the day-night rhythm to establish the relation between cell-sap pH and malate content. From the empirically determined pK values for malic acid in the cell sap, it was then possible to calculate the proportion of malate existing as the undissociated acid (H₂mal⁰) and in the anionic forms (Hmal^1– and mal^2–) for all times during the CAM rhythm. In leaf-slice experiments it has been found that the rate of malic-acid efflux increases exponentially with the malic-acid content of the tissue. This is shown to be related to the increasing amounts of H₂mal⁰ present at high malic-acid contents. At low malic-acid contents (<65 mol m^–3), when H₂mal⁰ is not present in significant amounts, efflux must be in the form of Hmal^–1 and/or mal^2–. At high malic-acid contents it is suggested that efflux occurs predominantly in the form of passive, noncatalyzed diffusion of H₂mal⁰ across the tonoplast by a lipid-solution mechanism. This is supported by the fact that the slope of the curve relating efflux to H₂mal⁰ concentration, when corrected for the presumed contributions from Hmal^1– and mal^2– transport and plotted on a log-log basis, approaches 1.0 at the highest malic-acid contents. Moreover, the permeability coefficient required to be consistent with such a mechanism is similar to that estimated from a Collander plot, using the partition coefficient of malic acid between ether and water. We suggest that may be important in determining the maximum amounts of malic acid that can be accumulated during the CAM rhythm.     

Ca Fluxes and Membrane Potentials in Nitella translucens

The concentrations of Ca have been measured in the flowing cytoplasmand the vacuole of the single cells of Nitella translucens,the cells being immersed in an artificial pond Water (composition:NaCl, 1.0 mM; KCl, 0.1 mM; CaCl₂, 0. mM). In the flowing cytoplasmthe total concentration is 8 mM and in the vacuole 12 mM. Measurementsof the electrical potential differences across the plasmalemmaand tonoplast membranes show that the cytoplasm is at a potentialof —134 mV with respect to the bathing medium and —24mV with respect to the vacuole. An attempt has been made tomeasure the tracer fluxes of Ca and it is shown that the cellsare not in flux equilibrium. The influx is 0.046 µµmoles cm^–2 sec^–1; the efflux was too small to measurewith any degree of accuracy. The observed potential differences across both membranes arecompared with the Nernst potentials for Ca. This analysis showsthat Ca is not in electrochemical equilibrium across eithermembrane and that the driving forces on Ca are directed fromthe bathing medium and the vacuole into the cytoplasm. It issuggested that there is no necessity for a metabolically drivenCa pump at the plasmalemma because the low cytoplasmic Ca contentcould be due to the low permeability of the plasmalemma; theGoldman flux equation gives a value of P_Ca = 4.3x10^–8cm sec^–1. A Ca pump at the tonoplast appears to be necessaryto explain the steep electrochemical potential gradient fromthe vacuole to the cytoplasm. The efflux of Ca from the isolated cell wall has been measured.From these measurements it was possible to estimate the concentrationof indiffusible anions in the Donnan Free Space; the value obtainedwas 0.74 equiv. 1.^–1.     

Electrochemical investigation of active malic acid transport at the tonoplast into the vacuoles of the CAM plantKalanchoë daigremontiana

Summary The membrane potential of cells in leaf slices of the CAM plantKalanchoë daigremontiana Hamet et Perrier in the light and in the dark is –200 mV on the average; it is reversibly depolarized by the metabolic inhibitors FCCP (5×10^–6 m) and CN^– (5×10^–3 m); it shows the light-dependent transient oscillations ubiquitously observed in green cells; it is independent of the amount of malic acid accumulated in the cells (in a tested range between 30 and 140mm); and it is considerably hyperpolarized by the fungal toxin fusicoccin (30×10^–6 m). Fusicoccin inhibits nocturnal malic acid accumulation in intact isolated phyllodia of the CAM plantKalanchoë tubiflora (Harv.) Hamet but does not affect remobilization of malic acid during the day.Electrochemical gradients for the various ions resulting from dissociation of malic acid, i.e., H⁺, Hmal^– and mal^2–, were calculated using the Nernst equation. With a very wide range of assumptions on cytoplasmic pH and malate concentration results of calculations suggest uphill transport of H⁺ and Hmal^– from the cytoplasm into the vacuole, while mal^2– might be passively distributed at the tonoplast. On the basis of the present data the most likely mechanism of active malic acid accumulation in the vacuoles of CAM plants appears to be an active H⁺ transport at the tonoplast coupled with passive movement of mal^2– possibly mediated by a translocator (catalyzed diffusion), with subsequent formation of Hmal^– (2 H⁺+mal^2–H⁺+Hmal^–) at vacuolar pH's.     

Modulation by Bicarbonate of Catalytic and Regulatory Properties of C4Phosphoenolpyruvate Carboxylase from Amaranthus hypochondriacus: Desensitization to Malate and Glucose 6-Phosphate and Sensitization to Mg2+

The catalytic and regulatory properties of phosphoenolpyruvate(PEP) carboxylase (PEPC) are modulated remarkably by the increasein the level of bicarbonate in the assay medium. The activityof PEPC increased by two-fold as the concentration of bicarbonatewas raised from 0.05 to 10 mM. During this state, there wasonly marginal effect on K_m for PEP, while the affinity of PEPCto Mg²⁺ increased by >2 fold. In contrast, the sensitivityof PEPC to malate decreased with increasing concentration ofHCO₃^–. Similarly, the stimulation by glucose 6-phosphate(G-6-P) at optimal concentration (10 mM) of HCO₃^– wasmuch less than that at suboptimal concentration (0.05 mM). K₁for malate increased by about 3 fold and K_a for G-6-P risedby fourfold as bicarbonate concentration was rised from 0.05to 10 mM. These results suggest that HCO₃^– desensitizesPEPC to both malate and G-6-P. Further, these changes were manifestedin both dark- as well as light-forms of the enzyme. Similarresults were obtained with PEPC in leaf extracts or in purifiedform. We therefore propose that bicarbonate-induced changesare independent of phospho-rylation and possibly through a significantchange in the conformation of the enzyme. This is the firstdetailed report indicating marked modulation of regulatory andcatalytic properties of PEPC by bicarbonate, one of its substrate. (Received April 14, 1998; Accepted September 22, 1998)     

A 31P-NMR Study of the Uptake and Compartmentation of Manganese by Maize Roots

The uptake and compartmentation of manganese by maize roots,from solutions containing between 1 µM and 1 mM Mn²⁺,was monitored in vivo by ³¹P nuclear magnetic resonance (NMR)spectroscopy. Qualitatively, NMR provided a convenient methodfor observing the effects of pH, anoxia, metabolic inhibitors,and competition with magnesium on the uptake of manganese andthe resultshighlighted the role of the vacuole as a sink forMn²⁺. Quantitatively, it was established thatroot tissues couldmaintain a low concentration of free Mn²⁺ in the cytoplasm duringmanganese uptake and that there is a non-equilibrium distributionof Mn²⁺ between the cytoplasm and the vacuole. Typically exposureto Mn²⁺ in the range 10–100 µM resulted in a submicromolarpool of Mn²⁺ in the cytoplasm and a vacuolar pool of 10 µMand it was concluded that the movement of Mn²⁺ out of the cytoplasmmust be energy consuming. Overall the results draw attentionto the similarity between the subcellular distribution of manganeseand calcium and provide some support for the suggestion thatmanganese, like calcium, might have a control function in normalcells. Key words: Cytoplasm, intracellular compartmentation, manganese, ³¹P-NMR, vacuole     

Apoplastic Solute Concentrations of Organic Acids and Mineral Nutrients in the Leaves of Several Fagaceae

Ion chromatographic methods determined organic acids and mainnutrient minerals in the apoplastic solution from leaves ofseveral Fagaceae (Quercus ilex L., Quercus cerris L., Quercusvirgiliana (Ten.) Ten, and Fagus sylvatica L.). The anions oforganic acids found in high amounts (250 to 650 µM) werequinate, malate, and oxalate. Lactate, pyruvate, formate andacetate were detected in relatively low amounts with concentrationsbetween 20 and 200 µM. The total concentration of organicacids in the apoplastic sap ranged between 1.5 and 2 mM. Thetotal concentration of inorganic cations (K⁺, Mg²⁺, NH₄⁺, Ca²⁺,Na⁺) and anions (C1^–, NO₃^–, SO^2–₄ and PO^3–₄)in the apoplastic sap varied between 5 and 10 mM, and 0.35 and1.8 mM, respectively. We conclude that the concentration oforganic acid ions in the leaf apoplast depends mainly on theexchange with the leaf cells and is influenced by the electrochemicalgradient between the symplast and the apoplast in relation tothe water potential of the leaf. The determination of formateand acetate in the apoplastic compartment of leaves lend weightto the argument that the production of these acids by treesis a important emission source to the atmosphere. (Received June 9, 1998; Accepted April 8, 1999)     

Day-to-night variations of cytoplasmic pH in a crassulacean acid metabolism plant

Summary In crassulacean acid metabolism (CAM) large amounts of malic acid are redistributed between vacuole and cytoplasm in the course of night-to-day transitions. The corresponding changes of the cytoplasmic pH (pH_cyt) were monitored in mesophyll protoplasts from the CAM plantKalanchoe daigremontiana Hamet et Perrier by ratiometric fluorimetry with the fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein as a pH_cyt indicator. At the beginning of the light phase, pH_cyt was slightly alkaline (about 7.5). It dropped during midday by about 0.3 pH units before recovering again in the late-day-to-early-dark phase. In the physiological context the variation in pH_cyt may be a component of CAM regulation. Due to its pH sensitivity, phosphoenolpyruvate carboxylase appears as a likely target enzyme. From monitoring ΔpH_cyt in response to loading the cytoplasm with the weak acid salt K-acetate a cytoplasmic H⁺-buffer capacity in the order of 65 mM H+ per pH unit was estimated at a pH_cyt of about 7.5. With this value, an acid load of the cytoplasm by about 10 mM malic acid can be estimated as the cause of the observed drop in pH_cyt. A diurnal oscillation in pH_cyt and a quantitatively similar cytoplasmic malic acid is predicted from an established mathematical model which allows simulation of the CAM dynamics. The similarity of model predictions and experimental data supports the view put forward in this model that a phase transition of the tonoplast is an essential functional element in CAM dynamics.     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)