Adaptation of the Cyanobacterium Anabaena variabilis to Low CO(2) Concentration in Their Environment

The rate of adaptation of high CO₂ (5% v/v CO₂ in air)-grown Anabaena to a low level of CO₂ (0.05% v/v in air) was determined as a function of O₂ concentration. Exposure of cells to low (2.6%) O₂ concentration resulted in an extended lag in the adaptation to low CO₂ concentration. The rate of adaptation following the lag was not affected by the concentration of O₂. The length of the lag period is markedly affected by the O₂/CO₂ concentration ratio, indicating that the signal for adaptation to low CO₂ may be related to the relative rate of ribulose-1,5-bisphosphate carboxylase/oxygenase activities, rather than to CO₂ concentration proper. This suggestion is supported by the observed accumulation of phosphoglycolate following transfer of cells from high to low CO₂ concentration.     

Inorganic Carbon Transport and Fixation in Cells of Anabaena variabilis Adapted to Mixotrophic Conditions

The Cyanobacterium Anabaena variabilis ATCC 29413 grown at lowCO₂ concentration under mixotrophic conditions with fructoseshowed a repression in the ability to fix inoganic carbon. Thisrepression was not due to a diminution in the ability to transportexternal inorganic carbon but could be explained by a decreaseof two enzymatic activities involved in the assimilation ofinorganic carbon: carbonic anhydrase and Rubisco. Carbonic anhydraseactivity was close to 50% lower in mixotrophic than in autotrophiccells. Moreover growth under mixotrophic conditions reducedRubisco activity at all dissolved inorganic carbon concentrationsassayed (5–60 mM). Maximum Rubisco activity (V_max decreasedfrom µmol CO₂ mg protein^-1h^-1 in autotrophic cells to2.3 µmol CO₂ mg protein^-1h^-1 in mixotrophic cells. Nosignificant differences in K_m(C₁) between autotrophic and mixotrophiccells were however observed. The possible mechanisms involvedin the inhibition of Rubisco are discussed. (Received November 8, 1994; Accepted October 12, 1995)     

Photosynthetic Carbon Metabolism in Photoautotrophic Cell Suspension Cultures Grown at Low and High CO(2)

Photosynthetic carbon metabolism was characterized in four photoautotrophic cell suspension cultures. There was no apparent difference between two soybean (Glycine max) and one cotton (Gossypium hirsutum) cell line which required 5% CO₂ for growth, and a unique cotton cell line that grows at ambient CO₂ (660 microliters per liter). Photosynthetic characteristics in all four lines were more like C₃ mesophyll leaf cells than the cell suspension cultures previously studied. The pattern of ¹⁴C-labeling reflected the high ratio of ribulosebisphosphate carboxylase to phosphoenolpyruvate carboxylase activity and showed that CO₂ fixation occurred primarily by the C₃ pathway. Photorespiration occurred at 330 microliters per liter CO₂, 21% O₂ as indicated by the synthesis of high levels of ¹⁴C-labeled glycine and serine in a pulse-chase experiment and by oxygen inhibition of CO₂ fixation. Short-term CO₂ fixation in the presence and absence of carbonic anhydrase showed CO₂, not HCO₃⁻, to be the main source of inorganic carbon taken up by the low CO₂-requiring cotton cells. The cells did not have a CO₂-concentrating mechanism as indicated by silicone oil centrifugation experiments. Carbonic anhydrase was absent in the low CO₂-requiring cotton cells, present in the high CO₂-requiring soybean cell lines, and absent in other high CO₂ cell lines examined. Thus, the presence of carbonic anhydrase is not an essential requirement for photoautotrophy in cell suspension cultures which grow at either high or low CO₂ concentrations.     

Photodissimilation of Fructose to H(inf2) and CO(inf2) by a Dinitrogen-Fixing Cyanobacterium, Anabaena variabilis

The ability of cyanobacteria to serve as biocatalysts in the production of H(inf2) as a fuel and chemical feedstock was investigated with Anabaena variabilis. The results show that A. variabilis, when incubated under argon, dissimilated fructose to H(inf2) and CO(inf2) in a light-dependent reaction. The H(inf2) production had an obligate requirement for fructose and was heterocyst dependent, since NH(inf4)(sup+)-grown cultures lacking heterocysts failed to produce H(inf2). Differential inhibition studies with CO showed that nitrogenase is the main enzyme catalyzing the H(inf2) production. Net H(inf2) yield increased with increasing concentrations of fructose up to 10 mM in the medium. The average apparent conversion efficiency of fructose to H(inf2) (net H(inf2) produced/fructose removed from the medium) was about 10, although higher conversion efficiencies of 15 to 17 could be obtained during shorter periods and at optimum fructose concentrations. Under the same conditions, the ratio of CO(inf2) released to fructose removed from the medium was about 3.5, suggesting that only a fraction of the fructose carbon was completely oxidized to CO(inf2). Under conditions of carbon excess, which prevents H(inf2) uptake, the maximum ratio of H(inf2) to CO(inf2) was found to be 3.0. This is higher than the expected value of 2.0, indicating that water was also a source of reductant in this fructose-mediated H(inf2) production. Inhibition of H(inf2) evolution by 3-(3,4-dichlorophenyl)-1,1-dimethylurea confirmed a role for photosystem II in this process. The rate of H(inf2) production by A. variabilis SA1 was 46 ml h(sup-1) g (dry weight)(sup-1). This high rate was maintained for over 15 days. About 30% of this H(inf2) was derived from water (10 ml of H(inf2) h(sup-1) g [dry weight](sup-1)). These results show that filamentous, heterocystous cyanobacteria can serve as biocatalysts in the high-efficiency conversion of biomass-derived sugars to H(inf2) as a fuel source while simultaneously dissimilating water to H(inf2).     

Involvement of a Primary Electrogenic Pump in the Mechanism for HCO(3) Uptake by the Cyanobacterium Anabaena variabilis

The response of the membrane potential to HCO₃⁻ supply has been studied in the cyanobacterium Anabaena variabilis strain M-3 under various conditions. Changes in potential were followed with the aid of the lipophilic cation tetraphenyl phosphonium bromide.     

Nature of the Inorganic Carbon Species Actively Taken Up by the Cyanobacterium Anabaena variabilis

The nature of the inorganic carbon (C_i) species actively taken up by cyanobacteria CO₂ or HCO₃⁻ has been investigated. The kinetics of CO₂ uptake, as well as that of HCO₃⁻ uptake, indicated the involvement of a saturable process. The apparent affinity of the uptake mechanism for CO₂ was higher than that for HCO₃⁻. Though the calculated V_max was the same in both cases, the maximum rate of uptake actually observed was higher when HCO₃⁻ was supplied. C_i uptake was far more sensitive to the carbonic anhydrase inhibitor ethoxyzolamide when CO₂ was the species supplied. Observations of photosynthetic rate as a function of intracellular C_i level (following supply of CO₂ or HCO₃⁻ for 5 seconds) led to the inference that HCO₃⁻ is the species which arrives at the inner membrane surface, regardless of the species supplied. When the two species were supplied simultaneously, mutual inhibition of uptake was observed.

On the basis of these and other results, a model is proposed postulating that a carboic anhydrase-like subunit of the C_i transport apparatus binds CO₂ and releases HCO₃⁻ at or near a membrane porter. The latter transports HCO₃⁻ ions to the cell interior.

     

Light-Induced Proton Release by the Cyanobacterium Anabaena variabilis: Dependence on CO(2) and Na

Light-induced acidification by the cyanobacterium Anabaena variabilis is biphasic (a fast phase I and slow phase II) and shown to be sodium-dependent with an optimum concentration of 40 to 60 millimolar Na⁺. Cells grown under low CO₂ concentrations at pH 9 (i.e. mainly HCO₃⁻ present in the medium) exhibited the slow phase II of proton efflux only, while cells grown under low CO₂ concentrations at pH 6.3 (i.e. CO₂ and HCO₃⁻ present) exhibited both phases. Light-induced proton release of phase I was dependent on inorganic carbon available in the bathing medium with an apparent K_m for CO₂ of 20 to 70 micromolar. As was concluded from the CO₂ dependence of acidification measured at different pH of the bathing medium, bicarbonate inhibited phase-I acidification noncompetetively. Acidification was inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Apparently, acidification of phase I is due to a light-dependent uptake of CO₂ being converted to HCO₃⁻ by a carbonic anhydrase-like function of the HCO₃⁻-transport system (M Volokita, D Zenvirth, A Kaplan, L Reinhold 1984 Plant Physiol 76: 599-602) before or during entering the cell, thus releasing one proton per CO₂ converted to HCO₃⁻.     

H2O2-Induced Inhibition of Photosynthetic O2 Evolution by Anabaena variabilis Cells

Hydrogen peroxide inhibits photosynthetic O2 evolution. It has been shown that H2O2 destroys the function of the oxygen-evolving complex (OEC) in some chloroplast and Photosystem (PS) II preparations causing release of manganese from the OEC. In other preparations, H2O2 did not cause or caused only insignificant release of manganese. In this work, we tested the effect of H2O2 on the photosynthetic electron transfer and the state of OEC manganese in a native system (intact cells of the cyanobacterium Anabaena variabilis). According to EPR spectroscopy data, H2O2 caused an increase in the level of photooxidation of P700, the reaction centers of PS I, and decreased the rate of their subsequent reduction in the dark by a factor larger than four. Combined effect of H2O2, CN-, and EDTA caused more than eight- to ninefold suppression of the dark reduction of P700+. EPR spectroscopy revealed that the content of free (or loosely bound) Mn2+ in washed cyanobacterial cells was ~20% of the total manganese pool. This content remained unchanged upon the addition of CN- and increased to 25-30% after addition of H2O2. The content of the total manganese decreased to 35% after the treatment of the cells with EDTA. The level of the H2O2-induced release of manganese increased after the treatment of the cells with EDTA. Incubation of cells with H2O2 for 2 h had no effect on the absorption spectra of the photosynthetic pigments. More prolonged incubation with H2O2 (20 h) brought about degradation of phycobilins and chlorophyll a and lysis of cells. Thus, H2O2 causes extraction of manganese from cyanobacterial cells, inhibits the OEC activity and photosynthetic electron transfer, and leads to the destruction of the photosynthetic apparatus. H2O2 is unable to serve as a physiological electron donor in photosynthesis.     

Quantification of the Contribution of CO2, HCO3-, and External Carbonic Anhydrase to Photosynthesis at Low Dissolved Inorganic Carbon in Chlorella saccharophila

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.     

Lag phase of CO2-dependent O2 evolution by illuminated Anabaena variabilis cells.

The steady-state rate of CO2-dependent O2 evolution by Anabaena variabilis cells in response to illumination was established after a lag phase. The lag phase was shortened (1) in cells incubated with glucose as an oxidizable substrate and (2) upon an increase in light intensity. The lag phase was absent during electron transfer from H2O to p-benzoquinone (in combination with ferricyanide) involving Photosystem II. A lag was observed during electron transfer from H2O to methyl viologen involving Photosystems II and I, but not for electron transfer from N,N,N',N'-tetramethyl-p-phenylenediamine (in combination with ascorbate) to methyl viologen involving only Photosystem I. The lag phases of the light-induced H2O --> CO2 and H2O --> methyl viologen electron transfer reactions showed the same temperature dependences at 10-30 degrees C. The lag was prevented by 3-(3,4-dichlorophenyl)-1,1-dimethylurea at concentrations that caused partial inhibition of photosynthetic O2 evolution. Retardation of cell respiration by a combination of CN- and benzylhydroxamate shortened the lag phase of the H2O --> methyl viologen electron transfer. It is concluded that the lag phase is associated with the electron transfer step between Photosystem II and Photosystem I common for the photosynthetic and respiratory chains and is due to the stimulation of cell respiration during the initial period of illumination as a consequence of an increase in the reduced plastoquinone pool and to subsequent retardation of respiration resulting from the transition of the electron transfer chain to the competitive pathway involving Photosystem I.     

Changes Induced by Low Oxygen Concentration in Photosynthetic and Respiratory CO2 Exchange in Phosphate-Deficient Bean Leaves

Effect of phosphorus deficiency on photosynthetic and respiratory CO₂ exchanges were analysed in primary leaves of 2-week-old bean (Phaseolus vulgaris L. cv. Golden Saxa) plants under non-photorespiratory (2 % O₂) and photorespiratory (21 % O₂) conditions. Low P decreased maximum net photosynthetic rate (P_Nmax) and increased the time necessary to reach it. In the leaves of P-deficient plants the relative decrease of P_Nmax at 2 % O₂ was larger than at 21 % O₂. The results suggested the influence of photorespiration in the cellular turnover of phosphates.     

Simultaneous Transport of CO(2) and HCO(3) by the Cyanobacterium Synechococcus UTEX 625

A mass spectrometer was used to simultaneously follow the time course of photosynthetic O₂ evolution and CO₂ depletion of the medium by cells of the cyanobacterium Synechococcus leopoliensis UTEX 625. Analysis of the data indicated that both CO₂ and HCO₃⁻ were simultaneously and continuously transported by the cells as a source of substrate for photosynthesis. Initiation of HCO₃⁻ transport by Na⁺ addition had no effect on ongoing CO₂ transport. This result is interpreted to indicate that the CO₂ and HCO₃⁻ transport systems are separate and distinctly different transport systems. Measurement of CO₂-dependent photosynthesis indicated that CO₂ uptake involved active transport and that diffusion played only a minor role in CO₂ acquisition in cyanobacteria.     

Carbon Oxysulfide Is an Inhibitor of Both CO(2) and HCO(3) Uptake in the Cyanobacterium Synechococcus PCC7942

Carbon oxysulfide (COS) was reinvestigated as an inhibitor of active inorganic carbon transport in cells of Synechococcus PCC7942 adapted to growth at low inorganic carbon. COS inhibited both CO₂ and HCO₃⁻ transport processes in a reversible (in the short term) and mixed competitive manner. The inhibition of COS was established using both silicone oil centrifugation experiments and O₂-evolution studies. The K_i for COS inhibition was 29 micromolar for CO₂ transport and 110 micromolar for HCO₃⁻ transport. These results support a model of inorganic carbon transport with a central CO₂ pump and an inducible HCO₃⁻ utilizing accessory protein which supplies CO₂ to the primary pump.     

Photoinhibition in Spirulina platensis: Response of Photosynthesis and HCO3- Uptake Capability to CO2 Depleted Conditions

The apparent photosynthetic affinity-for inorganic carbon inSpirulina is severely reduced by exposing the cells to CO₂-depletedconditions in the light. The extent of reduction depends onthe length of exposure, the light intensity, and the O₂ concentration.The photosynthetic dependence on inorganic carbon (C_Inorg) concentration,however, is similar at 3% O₂ and at 25% O₂ but in the presenceof catalase. The amount of O₂ released following the additionof catalase to the cell suspension also depends on the O₂ andCO₂ concentrations. The addition of H₂O₂ either to the cellsuspension or by a treatment with methyl viologen caused a severeinhibition of the capability of the cells to accumulate inorganiccarbon internally. It is suggested that the reduction of thephotosynthetic apparent affinity to C_Inorg upon subjecting thecells to photoinhibitory conditions is caused by an alterationof their ability to accumulate C_Inorg due to the accumulationof H₂O₂.     

C3 and CAM Photosynthetic Characteristics of the Submerged Aquatic Macrophyte Littorella uniflora: Regulation of Leaf Internal CO2 Supply in Response to Variation in Rooting Substrate Inorganic Carbon Concentration

The relationships between CO₂ concentrating mechanisms, photosyntheticefficiency and inorganic carbon supply have been investigatedfor the aquatic macrophyte Littorella uniflora. Plants wereobtained from Esthwaite Water or a local reservoir, with thelatter plants transplanted into a range of sediment types toalter CO₂ supply around the roots. Free CO₂ in sediment-interstitial-waterranged from 1–01 mol m^–3 (Esthwaite), 0.79 mol m^–3(peat), 0.32 mol m^–3 (silt) and 0–17 mol m^–3(sand), with plants maintained under PAR of 40 µmol m^–2s^–1. A comparison of gross morphology of plants maintained underthese conditions showed that the peat-grown plants with highsediment CO₂ had larger leaf fresh weight (0–69 g) andtotal surface area (223 cm² g^–1 fr. wt. including lacunalsurface area) than the sand-grown plants (0.21 g and 196 cm²g^–1 fr. wt. respectively). Root fresh weights were similarfor all treatments. In contrast, leaf internal CO₂ concentration[CO₂], was highest in the sand-grown plants (2–69 molm^–3, corresponding to 6.5% CO₂ in air) and lowest inthe Esthwaite plants (1–08 mol m^–3). Expressionof CAM in transplants was also greatest in the low CO₂ regime,with H⁺ (measured as dawn-dusk titratable acidity) of 50µmolg^– fr. wt., similar to Esthwaite plants in natural sediment.Assuming typical CAM stoichiometry, decarboxylation of malatecould account largely for the measured [CO₂]₁ and would makea major contribution to daytime CO₂ fixation in vivo. A range of leaf sections (0–2, 1–0, 5–0 and17–0 mm) was used to evaluate diffusion limitation andto select a suitable size for comparative studies of photosyntheticO₂ evolution. The longer leaf sections (17.0 mm), which weresealed and included the leaf tip, were diffusion-limited witha linear response to incremental addition of CO₂ and 1–0mol m^–3 exogenous CO₂ was required to saturate photosynthesis.Shorter leaf sections were less diffusion-limited, with thegreatest photosynthetic capacity (36 µmol O₂ g^–1 fr. wt. h^–1) obtainedfrom the 1.0 mm size and were not infiltrated by the incubatingmedium. Comparative studies with 1.0 mm sections from plants grown inthe different sediment types revealed that the photosyntheticcapacity of the sand-grown plants was greatest (45 µmolO₂ g^–1 fr. wt. h^–1) with a K_0.5 of 80 mmol m^–3.In terms of light response, saturation of photosynthesis intissue slices occurred at 850–1000 µmol m^–2s^–1 although light compensation points (6–11 µmolm^–2s^–1) and chlorophyll a: b ratios (1.3) were low.While CO₂ and PAR responses were obtained using varying numbersof sections with a constant fresh weight, the relationshipsbetween photosynthetic capacity and CO₂ supply or PAR were maintainedwhen the data were expressed on a chlorophyll basis. It is concludedthat under low PAR, CO₂ concentrating mechanisms interact inintact plants to maintain saturating CO₂ levels within leaflacunae, although the responses of the various components ofCO₂ supply to PAR require further investigation. Key words: Key words-Uttorella uniflora, internal CO₂ concentration, crassulacean acid metabolism, root inorganic carbon supply, CO₂ concentrating mechanism     

Involvement of Photosynthetic Carbon Reduction Cycle Intermediates in CO(2) Fixation and O(2) Evolution by Isolated Chloroplasts

The photosynthetic carbon reduction cycle intermediates can be divided into three classes according to their effects on the rate of photosynthetic CO₂ evolution by whole spinach (Spinacia oleracea) chloroplasts and on their ability to affect reversal of certain inhibitors (nigericin, arsenate, arsenite, iodoacetate, antimycin A) of photosynthesis: class I (maximal): fructose 1, 6-diphosphate, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, ribose-5-phosphate; class 2 (slight): glucose 6-phosphate, fructose 6-phosphate, ribulose-1, 5-diphosphate; class 3 (variable): glycerate 3-phosphate. While class 1 compounds influence the photosynthetic rate, they do not lower the Michaelis constant of the chloroplast for bicarbonate or affect strongly other photosynthetic properties such as the isotopic distribution pattern. It was concluded that the class 1 compounds influence the chloroplast by not only supplying components to the carbon cycle but also by activating or stabilizing a structural component of the chloroplast.     

Ammonium (methylammonium) transport by heterocysts and vegetative cells of Anabaena variabilis

Transport of the ammonium analogue [(14)C]methylammonium was similar in non-growing, fully differentiated heterocysts as compared to vegetative, multiplying cells of the filamentous cyanobacterium Anabaena variabilis. NH(4)(+) inhibited uptake into the cells and released accumulated methylammonium from the cells. These observations suggest that the main function of ammonium transport in heterocysts may not be NH(4)(+) acquisition but cyclic retention of ammonia produced by nitrogenase.     

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (II. Evidence for Induction in Response to External CO2 Concentration)

The critical species and concentrations of dissolved inorganic carbon (DIC) required for the induction of DIC transport during adaptation to low CO2 were determined for the green alga Chlorella ellipsoidea. The concentration of dissolved CO2 needed for the induction of both CO2 and HCO3- transport was independent of pH during adaptation, whereas the total DIC concentration required increased at alkaline pH. At pH 7.5, the minimum equilibrium DIC concentration at which high CO2 characteristics were maintained, i.e. transport was repressed, was 2100 [mu]M, whereas the maximum equilibrium DIC concentration below which DIC transport was fully induced (DICIND) was 500 [mu]M. Intracellular DIC concentration during adaptation to DICIND decreased temporarily after 2 h to 60% of the maximum level but recovered after 3 h of adaptation. After 3 h of adaptation to DICIND, cells exhibited maximum O2 evolution rate at DICIND. When cells partially adapted to DICIND were returned to high CO2, there was an immediate halt to the induction of transport and a gradual decrease in transport capacity over 23 h. The capacity for the induction of transport was unaffected by the absence of light. These results indicate that changes in the internal DIC pool during adaptation to low CO2 do not trigger the induction of DIC transport and that the induction is not light dependent. Induction of DIC transport in C. ellipsoidea appears to occur in response to the continuous exposure of cells to a critical CO2 concentration in the external medium.