Inorganic Carbon Accumulation Stimulates Linear Electron Flow to Artificial Electron Acceptors of Photosystem I in Air-Grown Cells of the Cyanobacterium Synechococcus UTEX 625.

The effect of inorganic carbon (Ci) transport and accumulation on photosynthetic electron transport was studied in air-grown cells of the cyanobacterium Synechococcus UTEX 625. When the cells were depleted of Ci, linear photosynthetic electron flow was almost completely inhibited in the presence of the photosystem I (PSI) acceptor N,N-dimethyl-p-nitrosoaniline (PNDA). The addition of Ci to these cells, in which CO2 fixation was inhibited with glycolaldehyde, greatly stimulated linear electron flow and resulted in increased levels of photochemical quenching and O2 evolution. In aerobic conditions substantial quenching resulted from methyl viologen (MV) addition and further quenching was not observed upon the addition of Ci. In anaerobic conditions MV addition did not result in quenching until Ci was added. Intracellular Ci pools were formed when MV was present in aerobic or anaerobic conditions or PNDA was present in aerobic conditions. There was no inhibitory effect of Ci depletion on electron flow to 2,6-dimethylbenzoquinone and oxidized diaminodurene, which accept electrons from photosystem II. The degree of stimulation of PNDA-dependent O2 evolution varied with the Ci concentration. The extracellular Ci, concentration required for a half-maximum rate (K1/2) was 3.8 [mu]M and the intracellular K1/2 was 1.4 mM for the stimulation of PNDA reduction. These values agreed closely with the K1/2 values of extracellular and intracellular Ci for O2 photoreduction. Linear electron flow to artificial electron acceptors of PSI was enhanced by intracellular Ci, which appeared to exert an effect on PSI or on the intersystem electron transport chain.

Inorganic Carbon-Stimulated O2 Photoreduction Is Suppressed by NO2- Assimilation in Air-Grown Cells of Synechococcus UTEX 625.

The effect of NO2- assimilation on O2 exchange and CO2 fixation of the cyanobacterium, Synechococcus UTEX 625, was studied mass spectrometrically. Upon addition of 1 mM inorganic carbon to the medium, inorganic carbon pools developed and accelerated O2 photoreduction 5-fold when CO2 fixation was inhibited. During steady-state photosynthesis at saturating light, O2 uptake represented 32% of O2 evolution and balanced that portion of O2 evolution that could not be accounted for by CO2 fixation. Under these conditions, NO2- assimilation reduced O2 uptake by 59% but had no influence on CO2 fixation. NO2- assimilation decreased both CO2 fixation and O2 photoreduction at low light and and increased net O2 evolution at all light intensities. The increase in net O2 evolution observed during simultaneous assimilation of carbon and nitrogen over carbon alone was due to a suppression of O2 photoreduction by NO2- assimilation. When CO2 fixation was precluded, NO2- assimilation inhibited O2 photoreduction and stimulated O2 evolution. When the electron supply was limiting (low light), competition among O2, CO2, and NO2- for electrons could be observed, but when the electron supply was not limiting (saturating light), O2 photoreduction and/or NO2- reduction caused electron transport that was additive to that for maximum CO2 fixation.

Photosynthetic Nitrite Reduction as Influenced by the Internal Inorganic Carbon Pool in Air-Grown Cells of Synechococcus UTEX 625.

Photosynthetic reduction of NO2- was studied in air-grown cells of a cyanobacterium, Synechococcus UTEX 625. Addition of NO2- resulted in significant amounts of chlorophyll a fluorescence quenching both in the absence and presence of CO2, fixation inhibitors, glycolaldehyde or iodoacetamide. The degree of NO2- quenching was insensitive to the O2 concentration in the medium. Addition of 100 [mu]M inorganic carbon in the presence of glycolaldehyde and O2, leading to formation of the carbon pool within the cells, resulted in pronounced fluorescence quenching. Removal of O2 from the medium restored the fluorescence yield completely, and the subsequent addition of NO2- quenched 36% of the variable fluorescence. From the response to added 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the quenching by NO2- appeared to be photochemical quenching, and nonphotochemical quenching did not seem to be present. The reduction of NO2- observed on its addition to inorganic carbon-depleted cells remained uninfluenced by O2 or glycolaldehyde. The internal inorganic carbon pool in the cells stimulated NO2- reduction, both in the presence and absence of O2, by 4.8-fold. An increase in NO2- reduction by 0.5-fold was also observed in the presence of O2 during simultaneous assimilation of carbon and nitrogen in inorganic carbon-depleted cells. Contrary to this, under anaerobiosis, NO2- reduction was suppressed when carbon and nitrogen assimilation occurred together.

High Affinity Transport of CO(2) in the Cyanobacterium Synechococcus UTEX 625.

The active transport of CO(2) in Synechococcus UTEX 625 was measured by mass spectrometry under conditions that preclude HCO(3) (-) transport. The substrate concentration required to give one half the maximum rate for whole cell CO(2) transport was determined to be 0.4 +/- 0.2 micromolar (mean +/- standard deviation; n = 7) with a range between 0.2 and 0.66 micromolar. The maximum rates of CO(2) transport ranged between 400 and 735 micromoles per milligram of chlorophyll per hour with an average rate of 522 for seven experiments. This rate of transport was about three times greater than the dissolved inorganic carbon saturated rate of photosynthetic O(2) evolution observed under these conditions. The initial rate of chlorophyll a fluorescence quenching was highly correlated with the initial rate of CO(2) transport (correlation coefficient = 0.98) and could be used as an indirect method to detect CO(2) transport and calculate the substrate concentration required to give one half the maximum rate of transport. Little, if any, inhibition of CO(2) transport was caused by HCO(3) (-) or by Na(+)-dependent HCO(3) (-) transport. However, (12)CO(2) readily interfered with (13)CO(2) transport. CO(2) transport and Na(+)-dependent HCO(3) (-) transport are separate, independent processes and the high affinity CO(2) transporter is not only responsible for the initial transport of CO(2) into the cell but also for scavenging any CO(2) that may leak from the cell during ongoing photosynthesis.

Mass Spectrometric Measurement of Intracellular Carbonic Anhydrase Activity in High and Low C(i) Cells of Chlamydomonas: Studies Using O Exchange with C/O Labeled Bicarbonate.

By measuring (18)O exchange from doubly labeled CO(2) ((13)C(18)O(18)O), intracellular carbonic anhydrase activity was studied with protoplasts and chloroplasts isolated from Chlamydomonas reinhardtii grown either on air (low inorganic carbon [C(i)]) or air enriched with 5% CO(2) (high C(i)). Intact low C(i) protoplasts had a 10-fold higher carbonic anhydrase activity than did high C(i) protoplasts. Application of dextran-bound inhibitor and quaternary ammonium sulfanilamide, both known as membrane impermeable inhibitors of carbonic anhydrase, had no influence on the catalysis of (18)O exchange, indicating that cross-contamination with extracellular carbonic anhydrase was not responsible for the observed activity. This intracellular in vivo activity from protoplasts was inhibited by acetazolamide and ethoxyzolamide. Intracellular carbonic anhydrase activity was partly associated with intact chloroplasts isolated from high and low C(i) cells, and the latter had a sixfold greater rate of catalysis. The presence of dextran-bound inhibitor had no effect on chloroplast-associated carbonic anhydrase, whereas 150 micromolar ethoxyzolamide caused a 61 to 67% inhibition of activity. These results indicate that chloroplastic carbonic anhydrase was located within the plastid and that it was relatively insensitive to ethoxyzolamide. Carbonic anhydrase activity in crude homogenates of protoplasts and chloroplasts was about six times higher in the low C(i) than in high C(i) preparations. Further separation into soluble and insoluble fractions together with inhibitor studies revealed that there are at least two different forms of intracellular carbonic anhydrase. One enzyme, which was rather insoluble and relatively insensitive to ethoxyzolamide, is likely an intrachloroplastic carbonic anhydrase. The second carbonic anhydrase, which was soluble and sensitive to ethoxyzolamide, is most probably located in an extrachloroplastic compartment.

Glycolaldehyde Inhibits CO(2) Fixation in the Cyanobacterium Synechococcus UTEX 625 without Inhibiting the Accumulation of Inorganic Carbon or the Associated Quenching of Chlorophyll a Fluorescence.

When studying active CO(2) and HCO(3) (-) transport by cyanobacteria, it is often useful to be able to inhibit concomitant CO(2) fixation. We have found that glycolaldehyde was an efficient inhibitor of photosynthetic CO(2) fixation in Synechococcus UTEX 625. Glycolaldehyde did not inhibit inorganic carbon accumulation due to either active CO(2) or HCO(3) (-) transport. When glycolaldehyde (10 millimolar) was added to rapidly photosynthesizing cells, CO(2) fixation was stopped within 15 seconds. The quenching of chlorophyll a fluorescence remained high (

Selective and Reversible Inhibition of Active CO(2) Transport by Hydrogen Sulfide in a Cyanobacterium.

The active transport of CO(2) in the cyanobacterium Synechococcus UTEX 625 was inhibited by H(2)S. Treatment of the cells with up to 150 micromolar H(2)S + HS(-) at pH 8.0 had little effect on Na(+)-dependent HCO(3) (-) transport or photosynthetic O(2) evolution, but CO(2) transport was inhibited by more than 90%. CO(2) transport was restored when H(2)S was removed by flushing with N(2). At constant total H(2)S + HS(-) concentrations, inhibition of CO(2) transport increased as the ratio of H(2)S to HS(-) increased, suggesting a direct role for H(2)S in the inhibitory process. Hydrogen sulfide does not appear to serve as a substrate for transport. In the presence of H(2)S and Na(+) -dependent HCO(3) (-) transport, the extracellular CO(2) concentration rose considerably above its equilibrium level, but was maintained far below its equilibrium level in the absence of H(2)S. The inhibition of CO(2) transport, therefore, revealed an ongoing leakage from the cells of CO(2) which was derived from the intracellular dehydration of HCO(3) (-) which itself had been recently transported into the cells. Normally, leaked CO(2) is efficiently transported back into the cell by the CO(2) transport system, thus maintaining the extracellular CO(2) concentration near zero. It is suggested that CO(2) transport not only serves as a primary means of inorganic carbon acquisition for photosynthesis but also serves as a means of recovering CO(2) lost from the cell. A schematic model describing the relationship between the CO(2) and HCO(3) (-) transport systems is presented.

Use of Carbon Oxysulfide, a Structural Analog of CO(2), to Study Active CO(2) Transport in the Cyanobacterium Synechococcus UTEX 625.

Carbon oxysulfide (carbonyl sulfide, COS) is a close structural analog of CO(2). Although hydrolysis of COS (to CO(2) and H(2)S) does occur at alkaline pH (>9), at pH 8.0 the rate of hydrolysis is slow enough to allow investigation of COS as a possible substrate and inhibitor of the active CO(2) transport system of Synechococcus UTEX 625. A light-dependent uptake of COS was observed that was inhibited by CO(2) and the ATPase inhibitor diethylstilbestrol. The COS taken up by the cells could not be recovered when the lights were turned off or when acid was added. It was concluded that most of the COS taken up was hydrolyzed by intracellular carbonic anhydrase. The production of H(2)S was observed and COS removal from the medium was inhibited by ethoxyzolamide. Bovine erythrocyte carbonic anhydrase catalysed the stoichiometric hydrolysis of COS to H(2)S. The active transport of CO(2) was inhibited by COS in an apparently competitive manner. When Na(+)-dependent HCO(3) (-) transport was allowed in the presence of COS, the extracellular [CO(2)] rose considerably above the equilibrium level. This CO(2) appearing in the medium was derived from the dehydration of transported HCO(3) (-) and was leaked from the cells. In the presence of COS the return to the cells of this leaked CO(2) was inhibited. These results showed that the Na(+)-dependent HCO(3) (-) transport was not inhibited by COS, whereas active CO(2) transport was inhibited. When COS was removed by gassing with N(2), a normal pattern of CO(2) uptake was observed. The silicone fluid centrifugation method showed that COS (100 micromolar) had little effect upon the initial rate of HCO(3) (-) transport or CO(2) fixation. The steady state rate of CO(2) fixation was, however, inhibited about 50% in the presence of COS. This inhibition can be at least partially explained by the significant leakage of CO(2) from the cells that occurred when CO(2) uptake was inhibited by COS. Neither CS(2) nor N(2)O acted like COS. It is concluded that COS is an effective and selective inhibitor of active CO(2) transport.

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii.

Mass spectrometric measurements of dissolved free (13)CO(2) were used to monitor CO(2) uptake by air grown (low CO(2)) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the (13)CO(2) concentration in the medium to close to zero. Similar results were obtained with low CO(2) cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO(2) uptake revealed that the removal of CO(2) from the medium in the light was due to selective and active CO(2) transport rather than uptake of total dissolved inorganic carbon. In the light, low CO(2) cells and protoplasts incubated with carbonic anhydrase took up CO(2) at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO(2) uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO(2) cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO(2) compensation point. During the linear uptake of CO(2), low CO(2) cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O(2) evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O(2) evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO(2) uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO(2) had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O(2) evolution. These results show low CO(2) cells of Chlamydomonas are able to transport both CO(2) and HCO(3) (-) but CO(2) is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO(2) from the dehydration of HCO(3) (-).

Photorespiratory ammonia does not inhibit photosynthesis in glutamate synthase mutants of Arabidopsis.

Exposure of ferredoxin-dependent glutamate synthase (EC 1.4.7.1) mutants of Arabidopsis thaliana to photorespiratory conditions resulted in the accumulation of NH(4) (+) and the inhibition of photosynthesis. However, upon transfer from 2% O(2), 350 microliters per liter CO(2), to 21% O(2), 350 microliters per liter CO(2), net photosynthesis declined at a slower rate in methionine sulfoximine treated leaf discs relative to controls. The recovery of photosynthesis was also more rapid in MSO-treated leaf discs although ammonia levels were more than threefold higher. Photosynthesis in leaf discs treated with azaserine was inhibited more than controls when transferred to 21% O(2) and recovered less than controls when returned to 2% O(2) although NH(4) (+) levels were not significantly different. The results obtained are consistent with the view that the rapid inhibition of photosynthesis in the glutamate synthase mutants in photorespiratory conditions is not due to the accumulation of NH(4) (+) but rather to the depletion of amino donors for glyoxylate and the consequent effects of glyoxylate on the lack of return of carbon to the chloroplast.

Characterization of the na-requirement in cyanobacterial photosynthesis.

The Na(+) requirement for photosynthesis and its relationship to dissolved inorganic carbon (DIC) concentration and Li(+) concentration was examined in air-grown cells of the cyanobacterium Synechococcus leopoliensis UTEX 625 at pH 8. Analysis of the rate of photosynthesis (O(2) evolution) as a function of Na(+) concentration, at fixed DIC concentration, revealed two distinct regions to the response curve, for which half-saturation values for Na(+) (K((1/2))[Na(+)]) were calculated. The value of both the low and the high K((1/2))(Na(+)) was dependent upon extracellular DIC concentration. The low K((1/2))(Na(+)) decreased from 1000 micromolar at 5 micromolar DIC to 200 micromolar at 140 micromolar DIC whereas over the same DIC concentration range the high K((1/2))(Na(+)) decreased from 10 millimolar to 1 millimolar. The most significant increases in photosynthesis occurred in the 1 to 20 millimolar range. A fraction of total photosynthesis, however, was independent of added Na(+) and this fraction increased with increased DIC concentration. A number of factors were identified as contributing to the complexity of interaction between Na(+) and DIC concentration in the photosynthesis of Synechococcus. First, as revealed by transport studies and mass spectrometry, both CO(2) and HCO(3) (-) transport contributed to the intracellular supply of DIC and hence to photosynthesis. Second, both the CO(2) and HCO(3) (-) transport systems required Na(+), directly or indirectly, for full activity. However, micromolar levels of Na(+) were required for CO(2) transport while millimolar levels were required for HCO(3) (-) transport. These levels corresponded to those found for the low and high K((1/2))(Na(+)) for photosynthesis. Third, the contribution of each transport system to intracellular DIC was dependent on extracellular DIC concentration, where the contribution from CO(2) transport increased with increased DIC concentration relative to HCO(3) (-) transport. This change was reflected in a decrease in the Na(+) concentration required for maximum photosynthesis, in accord with the lower Na(+)-requirement for CO(2) transport. Lithium competitively inhibited Na(+)-stimulated photosynthesis by blocking the cells' ability to form an intracellular DIC pool through Na(+)-dependent HCO(3) (-) transport. Lithium had little effect on CO(2) transport and only a small effect on the size of the pool it generated. Thus, CO(2) transport did not require a functional HCO(3) (-) transport system for full activity. Based on these observations and the differential requirement for Na(+) in the CO(2) and HCO(3) (-) transport system, it was proposed that CO(2) and HCO(3) (-) were transported across the membrane by different transport systems.

Active Transport of Inorganic Carbon Increases the Rate of O(2) Photoreduction by the Cyanobacterium Synechococcus UTEX 625.

Chlorophyll a fluorescence of Synechococcus UTEX 625 was quenched during the transport of inorganic carbon, even when CO(2) fixation was inhibited by iodoacetamide. Measurements with a pulse modulation fluorometer showed that at least 75% of the quenching was due to oxidation of Q(a), the primary acceptor of photosystem II. Mass spectrometry revealed that transport of inorganic carbon increased the rate of O(2) photoreduction. Hence, O(2) could serve as an electron acceptor to allow oxidation of Q(a) even in the absence of CO(2) fixation.

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 O(2) evolution and CO(2) depletion of the medium by cells of the cyanobacterium Synechococcus leopoliensis UTEX 625. Analysis of the data indicated that both CO(2) and HCO(3) (-) were simultaneously and continuously transported by the cells as a source of substrate for photosynthesis. Initiation of HCO(3) (-) transport by Na(+) addition had no effect on ongoing CO(2) transport. This result is interpreted to indicate that the CO(2) and HCO(3) (-) transport systems are separate and distinctly different transport systems. Measurement of CO(2)-dependent photosynthesis indicated that CO(2) uptake involved active transport and that diffusion played only a minor role in CO(2) acquisition in cyanobacteria.

Regulation of o(2) concentration in soybean nodules observed by in situ spectroscopic measurement of leghemoglobin oxygenation.

A fiber optic spectrophotometric system was used to monitor the in vivo oxygenation of leghemoglobin in intact, attached soybean root nodules (Glycine max L. Merr. x USDA 16 Bradyrhizobium japonicum) which were flattened during development by growth in narrow, glass-walled cuvettes. When equilibrated at an external pO(2) of 20 kilopascals, leghemoglobin was 36.6 +/- 5.4% oxygenated, a value estimated to represent an infected cell O(2) concentration of 21.5 nanomolar. Increasing the external pO(2) from 20 to 25 kilopascals caused a rapid increase in leghemoglobin oxygenation, followed by a recovery to the initial level, all within 7.5 minutes. At 25 kilopascals O(2), the rates of H(2) and CO(2) evolution were similar to those at 20 kilopascals. Since respiration had not increased, the results support the proposal that nodules adapt to increased external pO(2) by regulating their resistance to O(2) diffusion.

Ammonia Production and Assimilation in Glutamate Synthase Mutants of Arabidopsis thaliana.

Ammonia production and assimilation(1) were examined in photorespiratory mutants of Arabidopsis thaliana L. lacking ferredoxin-dependent glutamate synthase (Fd-GluS) activity. Although photosynthesis was rapidly inhibited in these mutants in normal air, NH(4) (+) continued to accumulate. The accumulation of NH(4) (+) was also seen after an initial lag of 30 minutes in 2% O(2), 350 microliters per liter of CO(2) and after 90 minutes in 2% O(2), 900 microliters per liter of CO(2). The accumulation of NH(4) (+) in normal air and low O(2) was also associated with an increase in the total pool of amino acid-N and glutamine, and a decrease in the pools of glutamate, aspartate, alanine, and serine. Upon return to dark conditions, or to 21% O(2), 1% CO(2) in the light, the NH(4) (+) which had accumulated in the leaves was reassimilated into amino acids. The addition of methionine sulfoximine (MSO) resulted in higher accumulations of NH(4) (+) in glutamate synthase mutants and prevented the reassimilation of NH(4) (+) upon return to the dark. The addition of MSO also resulted in the accumulation of NH(4) (+) in glutamate synthase mutants in the light and in 21% O(2), 1% CO(2). These results indicate that glutamine synthetase is essential for the reassimilation of photorespiratory NH(4) (+) and for primary N assimilation in the leaves and strongly suggest that glutamate dehydrogenase plays only a minimal role in the assimilation of ammonia. Levels of NADH-dependent glutamate synthase (NADH-GluS) appear to be sufficient to account for the assimilation of NH(4) (+) by a GS/NADH-GluS cycle.

Dark Respiration during Photosynthesis in Wheat Leaf Slices.

The metabolism of [(14)C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-(14)C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO(2). An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after (14)CO(2) production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of (14)CO(2) over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO(2) as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO(2) was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the (14)CO(2) release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.

Chlorophyll a Fluorescence Yield as a Monitor of Both Active CO(2) and HCO(3) Transport by the Cyanobacterium Synechococcus UTEX 625.

Simultaneous measurements have been made of inorganic carbon accumulation (by mass spectrometry) and chlorophyll a fluorescence yield of the cyanobacterium Synechococcus UTEX 625. The accumulation of inorganic carbon by the cells was accompanied by a substantial quenching of chlorophyll a fluorescence. The quenching occurred even when CO(2) fixation was inhibited by iodoacetamide and whether the accumulation of inorganic carbon resulted from either active CO(2) or HCO(3) (-) transport. Measurement of chlorophyll a fluorescence yield of cyanobacteria may prove to be a rapid and convenient means of screening for mutants of inorganic carbon accumulation.

Active Transport of CO(2) by the Cyanobacterium Synechococcus UTEX 625 : Measurement by Mass Spectrometry.

Mass spectrometry has been used to confirm the presence of an active transport system for CO(2) in Synechococcus UTEX 625. Cells were incubated at pH 8.0 in 100 micromolar KHCO(3) in the absence of Na(+) (to prevent HCO(3) (-) transport). Upon illumination the cells rapidly removed almost all the free CO(2) from the medium. Addition of carbonic anhydrase revealed that the CO(2) depletion resulted from a selective uptake of CO(2), rather than a total uptake of all inorganic carbon species. CO(2) transport stopped rapidly (<3 seconds) when the light was turned off. Iodoacetamide (3.3 millimolar) completely inhibited CO(2) fixation but had little effect on CO(2) transport. In iodoacetamide poisoned cells, transport of CO(2) occurred against a concentration gradient of about 18,000 to 1. Transport of CO(2) was completely inhibited by 10 micromolar diethylstilbestrol, a membrane-bound ATPase inhibitor. Studies with DCMU and PSI light indicated that CO(2) transport was driven by ATP produced by cyclic or pseudocyclic photophosphorylation. Low concentrations of Na(+) (<100 microequivalents per liter), but not of K(+), stimulated CO(2) transport as much as 2.4-fold. Unlike Na(+)-dependent HCO(3) (-) transport, the transport of CO(2) was not inhibited by high concentrations (30 milliequivalents per liter) of Li(+). During illumination, the CO(2) concentration in the medium remained far below its equilibrium value for periods up to 15 minutes. This could only happen if CO(2) transport was continuously occurring at a rapid rate, since the continuing dehydration of HCO(3) (-) to CO(2) would rapidly raise the CO(2) concentration to its equilibrium value if transport ceased. Measurement of the rate of dissolved inorganic carbon accumulation under these conditions indicated that at least part of the continuing CO(2) transport was balanced by HCO(3) (-) efflux.

Na-Stimulation of Photosynthesis in the Cyanobacterium Synechococcus UTEX 625 Grown on High Levels of Inorganic Carbon.

Photosynthesis of washed cells of Synechococcus UTEX 625 grown on 5% CO(2) was markedly stimulated (647 +/- 50%) at pH 8.0 by the addition of low concentrations of NaCl (concentration required for half-maximal response, K((1/2),) = 18 micromolar). Studies with KCl and Na(2)SO(4) showed that the stimulation was due to Na(+). Photosynthesis at pH 6.1 was only slightly stimulated by Na(+). The response of photosynthesis at pH 8.0 to [Na(+)] was strongly sigmoidal for dissolved inorganic carbon ([DIC]

Evidence for Na-Independent HCO(3) Uptake by the Cyanobacterium Synechococcus leopoliensis.

At low levels of dissolved inorganic carbon (DIC) and alkaline pH the rate of photosynthesis by air-grown cells of Synechococcus leopoliensis (UTEX 625) was enhanced 7- to 10-fold by 20 millimolar Na(+). The rate of photosynthesis greatly exceeded the CO(2) supply rate and indicated that HCO(3) (-) was taken up by a Na(+)-dependent mechanism. In contrast, photosynthesis by Synechococcus grown in standing culture proceeded rapidly in the absence of Na(+) and exceeded the CO(2) supply rate by 8 to 45 times. The apparent photosynthetic affinity (K((1/2))) for DIC was high (6-40 micromolar) and was not markedly affected by Na(+) concentration, whereas with air-grown cells K((1/2)) (DIC) decreased by more than an order of magnitude in the presence of Na(+). Lithium, which inhibited Na(+)-dependent HCO(3) (-) uptake in air-grown cells, had little effect on Na(+)-independent HCO(3) (-) uptake by standing culture cells. A component of total HCO(3) (-) uptake in standing culture cells was also Na(+)-dependent with a K((1/2)) (Na(+)) of 4.8 millimolar and was inhibited by lithium. Analysis of (14)C-fixation during isotopic disequilibrium indicated that standing culture cells also possessed a Na(+)-independent CO(2) transport system. The conversion from Na(+)-independent to Na(+)-dependent HCO(3) (-) uptake was readily accomplished by transferring cells grown in standing to growth in cultures bubbled with air. These results demonstrated that the conditions experienced during growth influenced the mode by which Ssynechococcus acquired HCO(3) (-) for subsequent photosynthetic fixation.