Engineering photorespiration: current state and future possibilities.

Reduction of flux through photorespiration has been viewed as a major way to improve crop carbon fixation and yield since the energy-consuming reactions associated with this pathway were discovered. This view has been supported by the biomasses increases observed in model species that expressed artificial bypass reactions to photorespiration. Here, we present an overview about the major current attempts to reduce photorespiratory losses in crop species and provide suggestions for future research priorities.

Evolution of the biochemistry of the photorespiratory C2 cycle.

Oxygenic photosynthesis would not be possible without photorespiration in the present day O2 -rich atmosphere. It is now generally accepted that cyanobacteria-like prokaryotes first evolved oxygenic photosynthesis, which was later conveyed via endosymbiosis into a eukaryotic host, which then gave rise to the different groups of algae and streptophytes. For photosynthetic CO2 fixation, all these organisms use RubisCO, which catalyses both the carboxylation and the oxygenation of ribulose 1,5-bisphosphate. One of the reaction products of the oxygenase reaction, 2-phosphoglycolate (2PG), represents the starting point of the photorespiratory C2 cycle, which is considered largely responsible for recapturing organic carbon via conversion to the Calvin-Benson cycle (CBC) intermediate 3-phosphoglycerate, thereby detoxifying critical intermediates. Here we discuss possible scenarios for the evolution of this process toward the well-defined 2PG metabolism in extant plants. While the origin of the C2 cycle core enzymes can be clearly dated back towards the different endosymbiotic events, the evolutionary scenario that allowed the compartmentalised high flux photorespiratory cycle is uncertain, but probably occurred early during the algal radiation. The change in atmospheric CO2 /O2 ratios promoting the acquisition of different modes for inorganic carbon concentration mechanisms, as well as the evolutionary specialisation of peroxisomes, clearly had a dramatic impact on further aspects of land plant photorespiration.

Light-dependent bicarbonate uptake and CO2 efflux in the marine microalga Nannochloropsis gaditana.

Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO2 fluxes in cell suspensions using mass spectrometry. Addition of H13CO3- to cell suspensions in the dark caused a transient increase in the CO2 concentration in the medium far in excess of the equilibrium CO2 concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once equilibrium between the Ci species had been achieved, a CO2 efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO2 by active transport. Photosynthetic O2 evolution and the release CO2 in the dark depend on HCO3- uptake since both were inhibited by the anion exchange inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA inhibitor, markedly inhibited CO2 efflux in the dark, indicating that CO2 efflux was dependent upon the intracellular dehydration of HCO3-. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO2 and HCO3- and thus causes a subsequent release of CO2 to the external medium.

Ferric reduction by iron-limited Chlamydomonas cells interacts with both photosynthesis and respiration.

Iron limitation led to a large increase in extracellular ferricyanide (Fe[III]) reductase activity in cells of the green alga Chlamydomonas reinhardtii Dangeard. Mass-spectrometric measurement of gas exchange indicated that ferricyanide reduction in the dark resulted in a stimulation of respiratory CO2 production without affecting the rate of respiratory O2 consumption, consistent with the previously postulated activation of the oxidative pentose phosphate pathway in support of Fe(III) reduction by iron-limited Chlamydomonas cells (X. Xue et al., 1998, J. Phycol. 34: 939-944). At saturating irradiance, the rate of ferricyanide reduction was stimulated almost 3-fold, and this stimulation was inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea. Ferricyanide reduction during photosynthesis resulted in approximately a 50% inhibition of photosynthetic CO2 fixation at saturating irradiance, and almost 100% inhibition of CO2 fixation at sub-saturating irradiance. Photosynthesis by iron-sufficient cells was not affected by ferricyanide addition. Addition of 250 microM ferricyanide to iron-limited cells in which photosynthesis was inhibited (either by the presence of glycolaldehyde, or by maintaining the cells at the CO2 compensation point) resulted in a stimulation in the rate of gross photosynthetic O2 evolution. Chlorophyll a fluorescence measurements indicated a large increase in non-photochemical quenching during ferricyanide reduction in the light; the increase in nonphotochemical quenching was abolished by the addition of nigericin. These results suggest that reduction of extracellular ferricyanide (mediated at the plasma membrane) interacts with both photosynthesis and respiration, and that both of these processes contribute NADPH in the light.

Cloning, characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803.

A 3.3 kb HindIII restriction-digest DNA fragment was isolated from a Synechocystis sp. strain PCC6803 subgenomic plasmid library which strongly hybridized to a 349 bp fragment of the icfA (ccaA) gene from Synechococcus sp. strain PCC7942. DNA sequence analysis of the fragment revealed three open reading frames (ORFs), two of which potentially coded for pantothenate synthetase (ORF275) and cytidylate kinase (ORF230). The third, ORF274, was 825 bp in length, encoding a deduced polypeptide of 274 aa (Mr, 30747) that bears 55% sequence identity to the Synechococcus icfA (ccaA) translation product, a beta-type carbonic anhydrase (CA). A 932 bp EcoRI fragment containing ORF274 was subcloned into an expression vector and the construct was transformed into Escherichia coli for overexpression. Electrometric assays for CA activity revealed that whole cell extracts containing the recombinant protein significantly enhanced the rate of conversion of CO2 to HCO3- and that 98% of this catalytic activity was inhibited by ethoxyzolamide, a well-characterized CA inhibitor. Antisera derived against the overexpressed protein recognized a 30.7 kDa protein that was predominantly associated with the isolated carboxysome fraction from Synechocystis. These results provide molecular and physiological evidence for the identification of a ccaA homologue in Synechocystis PCC6803 that encodes a carboxysomal beta-type CA.

Ethoxyzolamide Differentially Inhibits CO2 Uptake and Na+-Independent and Na+-Dependent HCO3- Uptake in the Cyanobacterium Synechococcus sp. UTEX 625.

The effects of ethoxyzolamide (EZ), a carbonic anhydrase inhibitor, on the active CO2 and Na+-independent and Na+-dependent HCO3- transport systems of the unicellular cyanobacterium Synechococcus sp. UTEX 625 were examined. Measurements of transport and accumulation using radiochemical, fluorometric, and mass spectrometric assays indicated that active CO2 transport and active Na+-independent HCO3- transport were inhibited by EZ. However, Na+-independent HCO3- transport was about 1 order of magnitude more sensitive to EZ inhibition than was CO2 transport (50% inhibition = 12 [mu]M versus 80 [mu]M). The data suggest that both the active CO2 (G.D. Price, M.R. Badger [1989] Plant Physiol 89: 37-43) and the Na+ -independent HCO3 - transport systems possessed carbonic anhydrase-like activity as part of their mechanism of action. In contrast, Na+-dependent HCO3- transport was only partially (50% inhibition = 230 [mu]M) and noncompetitively inhibited by EZ. The collective evidence suggested that EZ inhibition of Na+ -dependent HCO3- transport was an indirect consequence of the action of EZ on the CO2 transport system, rather than a direct effect on HCO3- transport. A model is presented in which the core of the inorganic carbon translocating system is formed by Na+-dependent HCO3- transport and the CO2 transport system. It is argued that the Na+-independent HCO3 - utilizing system was not directly involved in translocation, but converted HCO3- to CO2 for use in CO2 transport.

Characterization of the non-photochemical quenching of chlorophyll fluorescence that occurs during the active accumulation of inorganic carbon in the cyanobacterium Synechococcus PCC 7942.

Previous work has shown that the maximum fluorescence yield from PS 2 of Synechococcus PCC 7942 occurs when the cells are at the CO2 compensation point. The addition of inorganic carbon (Ci), as CO2 or HCO3 (-), causes a lowering of the fluorescence yield due to both photochemical (qp) and non-photochemical (qN) quenching. In this paper, we characterize the qN that is induced by Ci addition to cells grown at high light intensities (500 µmol photons m(-2) s(-1)). The Ci-induced qN was considerably greater in these cells than in cells grown at low light intensities (50 µmol photons m(-2) s(-1)), when assayed at a white light (WL) intensity of 250 µmol photons m(-2) s(-1). In high-light grown cells we measured qN values as high as 70%, while in low-light grown cells the qN was about 16%. The qN was relieved when cells regained the CO2 compensation point, when cells were illuminated by supplemental far-red light (FRL) absorbed mainly by PS 1, or when cells were illuminated with increased WL intensities. These characteristics indicate that the qN was not a form of energy quenching (qE). Supplemental FRL illumination caused significant enhancement of photosynthetic O2 evolution that could be correlated with the changes in qp and qN. The increases in qp induced by Ci addition represent increases in the effective quantum yield of PS 2 due to increased levels of oxidized QA. The increase in qN induced by Ci represents a decrease in PS 2 activity related to decreases in the potential quantum yield. The lack of diagnostic changes in the 77 K fluorescence emission spectrum argue against qN being related to classical state transitions, in which the decrease in potential quantum yield of PS 2 is due either to a decrease in absorption cross-section or by increased 'spill-over' of excitation energy to PS 1. Both the Ci-induced qp (t 0.5<0.5 s) and qN (t 0.5≃1.6 s) were rapidly relieved by the addition of DCMU. The two time constants give further support for two separate quenching mechanisms. We have thus characterized a novel form of qN in cyanobacteria, not related to state transitions or energy quenching, which is induced by the addition of Ci to cells at the CO2-compensation point.

Monensin Inhibition of Na+-Dependent HCO3- Transport Distinguishes It from Na+-Independent HCO3- Transport and Provides Evidence for Na+/HCO3- Symport in the Cyanobacterium Synechococcus UTEX 625.

The effect of monensin, an ionophore that mediates Na+/H+ exchange, on the activity of the inorganic carbon transport systems of the cyanobacterium Synechococcus UTEX 625 was investigated using transport assays based on the measurement of chlorophyll a fluorescence emission or 14C uptake. In Synechococcus cells grown in standing culture at about 20 [mu]M CO2 + HCO3-, 50 [mu]M monensin transiently inhibited active CO2 and Na+-independent HCO3- transport, intracellular CO2 and HCO3- accumulation, and photosynthesis in the presence but not in the absence of 25 mM Na+. These activities returned to near-normal levels within 15 min. Transient inhibition was attributed to monensin-mediated intracellular alkalinization, whereas recovery may have been facilitated by cellular mechanisms involved in pH homeostasis or by monensin-mediated H+ uptake with concomitant K+ efflux. In air-grown cells grown at 200 [mu]M CO2 + HCO3- and standing culture cells, Na+-dependent HCO3- transport, intracellular HCO3- accumulation, and photosynthesis were also inhibited by monensin, but there was little recovery in activity over time. However, normal photosynthetic activity could be restored to air-grown cells by the addition of carbonic anhydrase, which increased the rate of CO2 supply to the cells. This observation indicated that of all the processes required to support photosynthesis only Na+-dependent HCO3- transport was significantly inhibited by monensin. Monensin-mediated dissipation of the Na+ chemical gradient between the medium and the cells largely accounted for the decline in the HCO3- accumulation ratio from 751 to 55. The two HCO3- transport systems were further distinguished in that Na+-dependent HCO3- transport was inhibited by Li+, whereas Na+-independent HCO3- transport was not. It is suggested that Na+-dependent HCO3- transport involves an Na+/HCO3- symport mechanism that is energized by the Na+ electrochemical potential.

Quenching of Chlorophyll a Fluorescence in Response to Na+-Dependent HCO3- Transport-Mediated Accumulation of Inorganic Carbon in the Cyanobacterium Synechococcus UTEX 625.

In the cyanobacterium Synechococcus UTEX 625, the yield of chlorophyll a fluorescence decreased in response to the transport-mediated accumulation of intracellular inorganic carbon (CO2 + HCO3- + CO32- = dissolved inorganic carbon [DIC]) and subsequently increased to a near-maximum level following photosynthetic depletion of the DIC pool. When DIC accumulation was mediated by the active Na+-dependent HCO3- transport system, the initial rate of fluorescence quenching was found to be highly correlated with the initial rate of H14CO3- transport (r = 0.96), and the extent of fluorescence quenching was correlated with the size of the internal DIC pool (r = 0.99). Na+-dependent HCO3- transport-mediated accumulation of DIC caused fluorescence quenching in either the presence or absence of the CO2 fixation inhibitor glycolaldehyde, indicating that quenching was not due simply to NADP+ reduction. The concentration of Na+ required to attain one-half the maximum rate of H14CO3- transport, at 20 [mu]M external HCO3-, declined from 9 to 1 mM as the external pH increased from 8 to 9.6. A similar pH dependency was observed when fluorescence quenching was used to determine the kinetic constants for HCO3- transport. In cells capable of Na+-dependent HCO3- transport, both the initial rate and extent of fluorescence quenching increased with increasing external HCO3-, saturating at about 150 [mu]M. In contrast Na+-independent HCO3- transport-mediated fluorescence quenching saturated at an HCO3- concentration of about 10 [mu]M. It was concluded that measurement of chlorophyll a fluorescence emission provided a convenient, but indirect, means of following Na+-dependent HCO3- transport and accumulation in Synechococcus.

Na-Independent HCO(3) Transport and Accumulation in the Cyanobacterium Synechococcus UTEX 625.

The active transport and intracellular accumulation of HCO(3) (-) by air-grown cells of the cyanobacterium Synechococcus UTEX 625 (PCC 6301) was strongly promoted by 25 millimolar Na(+).Na(+)-dependent HCO(3) (-) accumulation also resulted in a characteristic enhancement in the rate of photosynthetic O(2) evolution and CO(2) fixation. However, when Synechococcus was grown in standing culture, high rates of HCO(3) (-) transport and photosynthesis were observed in the absence of added Na(+). The internal HCO(3) (-) pool reached levels up to 50 millimolar, and an accumulation ratio as high as 970 was observed. Sodium enhanced HCO(3) (-) transport and accumulation in standing culture cells by about 25 to 30% compared with the five- to eightfold enhancement observed with air-grown cells. The ability of standing culture cells to utilize HCO(3) (-) from the medium in the absence of Na(+) was lost within 16 hours after transfer to air-grown culture and was reacquired during subsequent growth in standing culture. Studies using a mass spectrometer indicated that standing culture cells were also capable of active CO(2) transport involving a high-affinity transport system which was reversibly inhibited by H(2)S, as in the case for air-grown cells. The data are interpreted to indicate that Synechococcus possesses a constitutive CO(2) transport system, whereas Na(+)-dependent and Na(+)-independent HCO(3) (-) transport are inducible, depending upon the conditions of growth. Intracellular accumulation of HCO(3) (-) was always accompanied by a quenching of chlorophyll a fluorescence which was independent of CO(2) fixation. The extent of fluorescence quenching was highly dependent upon the size of the internal pool of HCO(3) (-) + CO(2). The pattern of fluorescence quenching observed in response to added HCO(3) (-) and Na(+) in air-grown and standing culture cells was highly characteristic for Na(+)-dependent and Na(+)-independent HCO(3) (-) accumulation. It was concluded that measurements of fluorescence quenching provide an indirect means for following HCO(3) (-) transport and the dynamics of intracellular HCO(3) (-) accumulation and dissipation.

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.

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) (-).

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.

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.

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.