Effect of Ammonia on Dark CO2 Fixation by Anabaena Cells Treated with Methionine Sulfoximine

Dark CO₂ fixation by Anabaena cylindrica was stimulated aboutthree-fold by the addition of NH₄Cl to the cells. The ¹⁴CO₂incorporation experiments showed that ¹⁴C is most rapidly incorporatedinto aspartate and then glutamine by adding NH4CI. Glutamineaccumulated predominantly after the addition of NH₄Cl showingthat NH₄ is incorporated into glutamine by glutamine synthetase.The stimulating effect of NH4Cl on CO₂ fixation and amino acidsynthesis was suppressed by methionine sulfoximine, an inhibitorof glutamine synthetase. It was suggested that dark CO₂ fixationwas stimulated by the action of glutamine synthesis which isenhanced by ammonia. (Received February 10, 1981; Accepted April 2, 1981)     

Effect of Ammonium on Dark-CO2 Fixation and on Cytosolic and Vacuolar pH Values in Eremosphaera viridis de Bary (Chlorococcales)

At constant external [CO₂], rates of dark-CO₂ fixation of theunicellular green alga Eremosphaera viridis were drasticallyincreased (up to 40-fold) by addition of ammonium (NH₃+ NH₄⁺)at external pH values (pH₀) between 6.0 and 8.0. The cytosolicpH was monitored under identical conditions by micro-pH-electrodemeasurements, and cytosolic and vacuolar pH by the ³¹P-NMR technique.Addition of ammonium (5.0 mol m^– pH₀ 7.0) caused a rapidand transient acidification of the cytosol during the first4 min. Thereafter, the cytosolic pH remained constant at itsoriginal value. A rather constant cytosolic pH was also confirmedby ³¹P-NMR measurements, which, in addition, indicated a slowalkalization of the vacuole (about 0.5 units within 30 min afteraddition of ammonium). Since the dramatic stimulation of dark-CO₂ fixation by ammoniumis not mediated by an alkalization of the cytosol, nor by directammonium effects on phosphoenolpyruvate carboxylase (PEPC, E.C.4.1.1.31 [EC] ), the role of vacuolar alkalization as a possible triggerfor the stimulation of PEP-carboxylase is discussed. Key words: Cytosolic pH, dark-CO₂ fixation, pH-regulation, vacuolar pH     

Effects of Ammonia on Respiration, Adenylate Levels, Amino Acid Synthesis and CO2 Fixation in Cells of Chlorella vulgaris 11h in Darkness

The effects of NH₄Cl on respiration, adenylate and free aminoacid levels as well as dark CO₂ fixation were investigated usingnitrogen-starved Chlorella vulgaris 11h cells with or withoutaddition of methionine sulfoximine (MSX), an inhibitor of glutaminesynthetase. Upon addition of NH₄Cl (1 mM) to the cells not treatedwith MSX, respiration was stimulated and the level of ATP droppedrapidly, while the levels of ADP and AMP increased. NH₄Cl alsostimulated amino acid synthesis, especially of glutamine, andmarkedly enhanced dark CO₂ fixation. Addition of NH₄Cl to MSX-treatedcells stimulated respiration and lowered the level of ATP, butdid not enhance glutamine synthesis and only slightly stimulateddark CO₂ fixation. ⁴On leave from Institute of Medical Science, Advance R &D Co. Minami-Hashimoto, Sagamihara, Kanagawa-ken 220, Japan (Received January 28, 1984; Accepted April 19, 1984)     

Effects of dark preincubation and chloramphenicol on blue light-induced CO2 incorporation in Chlorella cells

Chlorella cells incubated in the dark longer than 12 hr showedpronounced blue light-induced ¹⁴CO₂ fixation into aspartate,glutamate, malate and fumarate (blue light effect), whereasthose kept under continuous light showed only a slight bluelight effect, if any. 2) During dark incubation of Chlorellacells, phosphoenolpyruvate carboxylase activity and the capacityfor dark ¹⁴CO₂ fixation decreased significantly, whereas ribulose-1,5-diphosphatecarboxylase activity and the capacity for photosynthetic ¹⁴CO₂fixation (measured under illumination of white light at a highlight intensity) did not decrease. 3) In cells preincubatedin the dark, intracellular levels of phosphoenolpyruvate and3-phosphoglycerate determined during illumination with bluelight were practically equal to levels determined during illuminationwith red light. 4) The blue light effect was not observed incells incubated widi chloramphenicol, indicating that blue light-inducedprotein synthesis is involved in the mechanism of the effect. (Received April 9, 1971; )     

Antagonism Between Malate and Ethylene in the Regulation of Avena sativa Coleoptile Elongation

Etiolated Avena sativa L. coleoptile sections were used to determinethe influence of C₂H₄ on in vivo and in vitro rates of CO₂ fixation,and to measure the influence of various permutations of C₂H₄,CO₂, and malate on growth. Whereas 1 mM malate or 320 µI^-1 CO₂ stimulated growth by approximately 100 per cent, inhibitionof growth by 10-8 µ I_-1 C₂H₄ was substantial only in thepresence of malate or CO₂ The increase in growth rate in responseto these two agents was eliminated by the simultaneous applicationof C₂H₄. The in vivo rate of dark [¹⁴C]bicarbonate fixationand in vitro enzymic assays of fixation were not measurablyinhibited by C₂H₄. These results are discussed in the lightof evidence which indicates that CO₂-stimulated growth is mediatedby dark fixation. The data do not support the view that C₂H₄inhibition of growth results from an inhibition of fixation,but suggests that C₂H₄ may inhibit some step in the processby which malate stimulates growth.     

Non-Autotrophic CO2 Fixation and Drought Tolerance in Mosses

Cratoneuron filicinum, a drought-sensitive moss, and Tortularuralis, a drought-tolerant moss, fix CO₂ non-autotrophicallyat a rate of about 1.2 and 2.2 µmol h^–1 g^–1dry wt. respectively. During drying, T. ruralis fixes CO₂ atan undiminished rate until the tissue loses about 60% of theinitial fresh weight. Thereafter, CO₂ fixation rapidly declinesto zero. Dark CO₂ fixation by C.filicinum declines steadilyduring the dehydration period. On rehydration, dark CO₂ fixationis resumed immediately in T. ruralis but not in C.filicinum.When dried T. ruralis is equilibrated with an atmosphere ofnearly 100% relative humidity, its weight increases to about40% of the original fresh weight and dark CO₂ fixation resumesat a rate about 60% of the fresh moss. In C.filicinum thereis only a small increase in weight and little CO₂ fixation inthe dark. The non-autotrophically fixed carbon, in both mossesstudied, is incorporated into amino acids (more than 60% ofthe total, mainly into aspartate, alanine and glutamate) andorganic acids (less than 40% of the total, mainly into malate).It is suggested that on rehydration immediate availability ofNADPH, known to be produced by transhydrogenation from NADHduring dark CO₂ fixation, may be an important factor in therepair of drought-induced cellular damage by reductive biosynthesisof membrane components and other cellular constituents. Key words: Mosses, Dehydration, Rehydration, Dark CO₂ fixation, Amino acids, Organic acids, NADPH, Drought tolerance.     

Role of Carbonic Anhydrase in Photosynthesis of Blue-Green Alga (Cyanobacterium) Anabaena variabilis ATCC 29413

The affinity for NaHCO₃ (CO₂) in photosynthesis of Anabaenavariabilis ATCC 29413 was much higher in the cells grown underordinary air (low-CO₂ cells) than in those grown in air enrichedwith 2–4% CO₂ (high-CO₂ cells) (pH 8.0, 25?C). Ethoxyzolamide(50 µM) increased the K_m(NaHCO₃ in low-CO₂ cells aboutnine times (from 14.3 to 125), while the maximum rate of photosynthesisdecreased about 20%. When high-CO₂ cells were transferred tolow-CO₂ conditions, carbonic anhydrase (CA) activity increased,while K_m(NaHCO₃) in photosynthesis decreased from 140 to 30µM within about 5 h. The addition of CA to the suspensionof both high- and low-CO₂ cells enhanced the rates of photosyntheticO₂ evolution under CO₂-limiting conditions. The rate of ¹⁴CO₂fixation was much faster than that of H¹⁴CO₃^– fixation.The former reaction was greatly suppressed, while the latterwas enhanced by the addition of CA. These results indicate thatthe active species of inorganic carbon utilized for photosynthesiswas free CO₂ irrespective of the CO₂ concentration given duringgrowth. It is suggested that CA plays an active role in increasingthe affinity for CO₂ in photosynthesis of low-CO₂ cells of thisblue-green alga. (Received January 24, 1984; Accepted October 22, 1984)     

Accumulation and Utilization of Dissolved Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi

The mechanism for utilization of dissolved inorganic carbon(DIC) was investigated in the marine unicellular calcareousalga Emiliania huxleyi, grown with constant aeration. The apparentK_0.5 (DIC), the concentration of DIC which attains one-halfof the maximum velocity of apparent photosynthesis, for photosyntheticevolution of O₂, measured under saturating light, was 5.5 mM(55 µM for CO₂) at pH 8.0 and 25°C. The value of K_0.5was not affected by inhibitors of carbonic anhydrase (CA), andan electrometric assay of CA showed that the enzyme was notinvolved in photosynthesis in this alga. The rate of photosyntheticfixation of ¹⁴C-DIC into acid-stable products was about 20 timeshigher than that into CaCO₃, irrespective of the external concentrationof DIC. In short-term experiments, ¹⁴C-DIC was usually incorporatedinto the internal pool of DIC (IIC) to concentrations up to13 to 16 times higher than that of the external DIC. CO₂ addedexternally was utilized mainly for fixation of CO₂ and accumulationof IIC. By contrast, HCO^-₃ was utilized mainly for productionof CaCO₃ and accumulation of IIC. Incorporation of ¹⁴C intoIIC was partially suppressed by DCMU or in darkness but itstransfer to CaCO₃ was unaffected. These results suggest thataccumulation of IIC in this alga, even under ordinary circumstances,is only partially responsible for increasing the efficiencyof utilization of DIC by photosynthetic fixation but may bemost useful for the production of CaCO₃. (Hydroxyethylidene) bisphosphonic acid, an inhibitor of thegrowth of CaCO₃ crystals, completely suppressed production ofCaCO₃. The accumulation of IIC was also partially suppressed,but photosynthetic fixation of CO₂ was enhanced. In a pulse-chaseexperiment with ¹⁴CDIC, ¹⁴C incorporated into IIC and CaCO₃in darkness was transferred to acid-stable products of photosynthesisin the light. These results suggest that ¹⁴C-DIC in IIC andpre-formed CaCO₃ may be useful sources of carbon for fixationof CO₂. (Received July 2, 1993; Accepted January 10, 1994)     

The Effect of External pH on the Apparent CO2 Affinity of Chlorella saccharophila

The carbon dioxide compensation point of the unicellular greenalga, Chloretla saccharophila, was determined in aqueous mediumby a gas chromatographic method. Compensation points decreasedmarkedly from 63 cm³ m^–3 at an external pH of 4.0 to 3.2cm³ m^–3 at pH 8.0 and were not affected by the O₂ concentrationof the medium. The calculated CO² concentration required tosupport the half-maximum photosynthetic rate of the algal cellsranged from 6.0 mmol m_–3 at an external pH of 60 to 1.5mmol m^–3 at pH 8.0 and these values were not affectedby O₂ concentration. The K_m(CO₂) of nbulose-l,5-bisphosphatecarboxylase isolated from cells grown either at pH 4.0 or pH8.0 was determined to be 64 mmol m^–3. These results indicatethat loss of CO₂ by photorespiration does not occur in C. saccharophilacells at acid pH and the disparity between the apparent affinityfor CO₂ of the intact cells and that of the carboxylase indicatesthe operation of a ‘CO₂ concentrating mechanism’in this alga at acid pH. Key words: Acidophilic alga, bicarbonate transport, Chlorella saccharophila, compensation point, CO₂ affinity, PH, RuBP carboxylase     

Studies on Dark Fixation of Carbon Dioxide in Kalanchoe: II. Effect of Interaction of Photoperiodic Induction with Water Stress and Growth Retardants

In Kalanchoë blossfeldiana von Poellnitz cv. Tom Thumband cv. Feuer Blute interaction of CO₂ fixation with photoperiodicinduction and water stress was examined. It was found that TomThumb raised in dilute culture solution and kept in photoinductivecycles (8 h light and 16 h dark) flowered but failed to showa net dark CO₂ fixation. A net dark fixation was observed whenthe concentration of the culture solution was increased or plantswere sprayed with 300 or 750 mg 1^–1 CCC or 300 or 2000mg 1^–1 B-9. In a non-inductive photoperiod no net darkfixation was observed with these treatments although there wasa tendency for dark fixation to increase. Feuer Blute did not flower in inductive photoperiods when keptin half strength solution. It is suggested that in Tom Thumbboth photoperiodic induction and water stress are required forinitiation of net CO₂ dark fixation. In Feuer Blute where CAMis occurring normally photo-induction is sufficient to induceflowering. In half strength solution CO2 dark fixation is disturbedand floral induction also does not occur.     

Studies on Dark Fixation of Carbon Dioxide in Kalanchoe: I. Effect of Water Stress and Growth Retardants

Rooted cuttings of Kalanchoë blossfeldiana cv. Feuer Bluteand K. crenatum failed to show a net dark CO₂ fixation whenraised in dilute nutrient solution. Dark CO₂ fixation (CAM)in these plants was initiated either by increasing the soluteconcentration or lowering the water potential of the nutrientsolution by addition of mannitol (0.11 M and 0.25 M) and carbowax4000 (0.16 M and 0.3 M). Initiation was also brought about byspraying the leaves with B-9 (N,dimethylamino-succinamicacid,300mg1^–1) or by addition of CCC (2 chloroethyl trimethylammonium chloride, 300 or 750 mg1^–1) to the nutrient medium.Failure of CAM in dilute solution was suggested to be due tolack of accumulation of photosynthates in the leaves. Waterstress and growth retardants brought about reduction of monilizationand/or translocation thereby leading to accumulation of assimilatesin the leaves and to initiation of dark CO₂ fixation.     

Two Polypeptides Inducible by Low Levels of CO2 in Soluble Protein Fractions from Chlorella regularis Grown at Low or High pH

Previous studies suggested that certain protein(s) other thancarbonic anhydrase might play an important role in the facilitatedtransport of dissolved inorganic carbon (DIC) from the mediumto the site of CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenasein the unicellular green alga Chlorella regularis adapted tolow-CO₂ (ordinary air) conditions [Shiraiwa et al. (1991) Jpn.J. Phycol. 39: 355; Satoh and Shiraiwa (1992) Research in Photosynthesis,Vol. III, p. 779]. The proteins that might be involved in thisfacilitated transport of DIC were investigated by pulse-labelingof induced proteins with ³⁵S-sulfate during adaptation of cellsgrown under high-CO₂ conditions to low CO₂. Analysis by SDS-PAGErevealed that synthesis of two polypeptides, with molecularmasses of 98 and 24 kDa, respectively, was induced under low-CO₂conditions. The 24-kDa polypeptide was induced at pH 5.5 butnot at pH 8.0, whereas the 98-kDa polypeptide was induced atboth pH 5.5 and pH 8.0. The possible role of these polypeptidesin the facilitated transport of DIC in Chlorella regularis isdiscussed. (Received October 30, 1995; Accepted February 26, 1996)     

Stimulation of dark CO2 fixation by ammonia in isolated mesophyll cells of Papaver somniferum L.

Addition of 2 mM ammonium ion to isolated mesophyll cells ofPapaver somniferum resulted in a 3-fold or greater increasein their rate of dark ¹⁴C fixation, while even 0.1 mM NH₄⁺ nearlydoubled that rate. The most rapid increase in the labeling ofa metabolite occurred in aspartate, and was accompanied by adecrease in the steady-state level of labeled phosphoenolpyruvate.No change in labeled pyruvate level occurred, and alanine labelingdeclined. Ammonium ion addition had no effect on the respiratoryrate of these cells in the dark. We conclude that NH₄⁺ stimulatesphosphoenolpyruvate carboxylation in this system, but has nodetectable effect on the pyruvate kinase reaction in the dark.Our results are compared with earlier findings and the possibleregulatory function of ammonia is discussed. (Received July 23, 1979; )     

Characterization of the Type of Photosynthetic Carbon Dioxide Fixation in Jute (Corchorus olitorius L.)

Activities of photosynthetic and photorespiratory enzymes viz.,ribulose bisphosphate carboxylase, phosphoenol pyruvate carboxylaseand glycolate oxidase from jute (Corchorus olitorius L.; cv.JRO 632) leaves were compared with those from maize (C₄) andsunflower (C₃) leaves. The photosynthetic CO₂ fixation products,the release of ¹⁴CO₂ in light and dark following photosynthesisin ¹⁴CO₂, chlorophyll a: b ratio, gross leaf photosyntheticrate and dry matter production rate were also studied. The resultsshow that jute is a C₃ plant. Key words: Jute, Corchorus olitorius, C₃ photosynthesis     

Operation of the reductive pentose phosphate cycle daring the induction period of photosynthesis in Chlorella

When Chlorella oulgaris ll h cells grown in air containing 4%CO₂ (high-CO₂ cells) were given low concentrations of¹⁴CO₂ (<150ppm), the initial rate of photosynthetic ¹⁴CO₂ fixation wasvery low and linear ¹⁴CO₂ fixation was observed after an inductionperiod which lasted for ca. 45 min. No such induction period was observed when high-CO₂ cells weregiven high concentrations of ¹⁴CO₂ (10,000 ppm) or when IOW-CO₂cells were given either low or high concentrations of ¹⁴CO₂,supporting the observations by Briggs and Whittingham (l). However,irrespective of CO₂ concentrations during growth and of ¹⁴CO₂concentrations during the experiments, most of the ¹⁴C was incorporatedinto phosphate esters during the initial periods of photosynthetic¹⁴CO₂ fixation. These results are in sharp contrast to the reportby Graham and Whittingham (4). ¹ Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received June 30, 1979; )     

Carbon Dioxide Fixation by Guard Cell Protoplasts of Commelina communis

Biochemical studies of epidermal tissue may not reflect metabolismof the guard cells which represent less than 5% of the tissuevolume. Pure samples of guard cell protoplasts of Commelinacommunis were therefore used to investigate CO₂ fixation ratesand ¹⁴C-labelling patterns of metabolites in the light and thedark. Qualitatively, results were similar in most respects tothose obtained in a previous study (Schnabl, 1980) for guardcell protoplasts of Vicia faba. CO₂ fixation rates by guardcell protoplasts of C. communis were the same in the light andthe dark but about 50 times lower than the values Schnabl obtainedfor V.faba. The ¹⁴C-labelling pattern of metabolites in C. communiswas also similar in the light and the dark: over 60% of thetotal fixed was in malate with only 1% in sugar phosphates.Label was also detected in starch, aspartate, glutamate andcitrate but not in glycollate as previously recorded in V. fabaguard cell protoplasts. The results confirm the view that the reductive pentose phosphatepathway does not occur in guard cells of C. communis. Key words: CO₂ fixation, Guard cell protoplasts, Stomata     

Effect of leaf age on light and dark 14CO2 fixation in a CAM plant, Bryophyllum calycinum

Leaves of different ages from B. calycinum were exposed to ¹⁴CO₂in light during day and night. The labelling pattern on thechromatogram differed with leaf age. Young leaves had similarpatterns to those of C3 plants during both day and night. Matureleaves showed high incorporation of ¹⁴C into C4 acids, especiallyat night. In contrast, no significant difference with leaf agewas observed in the pattern of dark ¹⁴CO₂ fixation products.Study of the enzyme activity and the content of titratable acidat each leaf age suggested that high incorporation of ¹⁴C inC4 acids during the night was due to the simultaneous absorptionof CO₂ by both enzymes RuDPcarboxylase and PEPcarboxylase. (Received November 24, 1977; )     

Translocation Profiles of [11C] and [13N]-Labelled Metabolites after Assimilation of 11CO2 and [13N]-Labelled Ammonia Gas by Leaves of Helianthus annuus L. and Lupinus albus L.

Tracer amounts of atmospheric [¹³N]-Iabelled ammonia gas, wereabsorbed by leaves of Lupinus albus and Helianthus annuus inboth the light and the dark. Exogenous [¹³N]-ammonia was onlyabsorbed in the dark when the feeding occurred shortly aftera period of illumination and the tissue was not depleted ofits carbohydrate reserves (e.g. starch). Incorporation of the[¹³N]-ammonia appeared to occur via the leaf glutamine synthetase/glutamatesynthase (GS/GOGAT) cycle since 2.0 mol m^–3 MSX, an inhibitorof the GS reduced uptake in both the light and dark. Photosyntheticincorporation of ¹¹CO₂ was not affected by this treatment Therate of movement of [¹³N]-assimilates in the petiole of attachedleaves of Helianthus and Lupinus was similar to that of the¹¹Cl-photo assimilates. Export of both [¹³N] and [¹¹C]-Iabelledassimilates from the leaf and movement in the petiole in boththe light and the dark was inhibited by source leaf anoxia (i.e.nitrogen gas). Translocation was re-established at the samerate when the feed leaf was exposed to gas containing more than2% O₂ which permitted dark respiration to proceed. After aninitial feeding of either ¹¹CO₂ or [¹³N]-ammonia at ambient(21%) O₂ exposure of the source leaf to 2% O2, or 50% O² didnot alter the rates of translocation, indicating that changesin photosynthetic activity in the source leaf due to photorespiratoryactivity need not markedly alter, at least during the shortperiod, the loading and translocation of either [¹¹C ] or [¹³N]-labelledleaf products. Key words: Translocation, CO₂, NH₃, Leaves, Helianthus annuus, Lupinus albus     

Active Uptake of CO2 by the Diatom Navicula pelliculosa

Mass spectrometry has been used to investigate the transportof CO₂ in the freshwater diatom Navicula pelliculosa. The timecourseof CO₂ formation in the dark after addition of 100 mmol m^–3dissolved inorganic carbon (DIC) to cell suspensions showedthat no external carbonic anhydrase (CA) was present in thesecells. Upon illumination, cells pre-incubated at pH 75 with100 mmol m^–3 DIC, removed almost all free CO₂ from themedium at an initial rate of 285 µmol CO₂ mg^–1Chl h^–1. Equilibrium between HCO₃^– and CO₂ in themedium occurred rapidly upon addition of bovine CA, showingthat CO₂ depletion resulted from a selective uptake of CO₂ ratherthan an uptake of all inorganic carbon species. However, photosyntheticO₂ evolution rate remained constant after CO₂ had been depletedfrom the medium indicating that photosynthesis is sustainedprimarily by active HCO₃^– uptake. Treatment of cells with2-iodoacetamide (83 mol m^–3) completely inhibited CO₂fixation but had little effect on CO₂ transport since initialrates of CO₂ depletion were about 81% that of untreated cells.Transfer of iodoacetamide-treated cells to the dark caused arapid increase in the CO₂ concentration in the medium largelydue to the efflux of the unfixed intracellular DIC pool whichwas found to be about 194 times the concentration of that inthe external medium. These results indicate that Navicula pelliculosaactively takes up molecular CO₂ against a concentration gradientby a process distinct from HCO₃^– transport. Key words: Dissolved inorganic carbon, carbonic anhydrase, bicarbonate transport, CO₂ transport, mass spectrometry     

External and Internal CO2 Transport in Lemanea: Interactions with the Kinetics of Ribulose Bisphosphate Carboxylase

The rate of photosynthesis by the freshwater alga Lemanea mamillosais proportional to CO₂ concentration, virtually to the pointof saturation, and inversely proportional to the radius of thethallus. By contrast, the CO₂ response curve of very thin slicesof the thallus is a rectangular hyperbola with a (lower) halfsaturation concentration of 10 mmol m^–3. For the intactplant, the kinetics of CO₂ fixation are strongly masked by internalCO₂ transport limitations, although the maximum rate of photosynthesisis probably determined by the rate of supply of ribulose bisphosphate(RuBP). The flow of water over the alga becomes turbulent atwater velocities greater than about 90 mm s^–1 and thethallus stretches significantly at higher water velocities.In its natural habitat, therefore, the external unstirred layerwill be thin (< 10 µm) and the thallus will be stretched,leading to rapid external and increased internal rates of CO₂transport from the bulk solution. The estimated maximum rateof CO₂ transport is commensurate with the maximum rate of photosynthesis(i.e. the rate of supply of RuBP). Key words: Transport limitations, Kinetics of CO₂ fixation