Air-grown Cells Research Articles

Microoxic Niches within the Thylakoid Stroma of Air-Grown Chlamydomonas reinhardtii Protect [FeFe]-Hydrogenase and Support Hydrogen Production under Fully Aerobic Environment.

Photosynthetic hydrogen production in the microalga Chlamydomonas reinhardtii is catalyzed by two [FeFe]-hydrogenase isoforms, HydA1 and HydA2, both irreversibly inactivated upon a few seconds exposure to atmospheric oxygen. Until recently, it was thought that hydrogenase is not active in air-grown microalgal cells. In contrast, we show that the entire pool of cellular [FeFe]-hydrogenase remains active in air-grown cells due to efficient scavenging of oxygen. Using membrane inlet mass spectrometry, (18)O2 isotope, and various inhibitors, we were able to dissect the various oxygen uptake mechanisms. We found that both chlororespiration, catalyzed by plastid terminal oxidase, and Mehler reactions, catalyzed by photosystem I and Flavodiiron proteins, significantly contribute to oxygen uptake rate. This rate is considerably enhanced with increasing light, thus forming local anaerobic niches at the proximity of the stromal face of the thylakoid membrane. Furthermore, we found that in transition to high light, the hydrogen production rate is significantly enhanced for a short duration (100 s), thus indicating that [FeFe]-hydrogenase functions as an immediate sink for surplus electrons in aerobic as well as in anaerobic environments. In summary, we show that an anaerobic locality in the chloroplast preserves [FeFe]-hydrogenase activity and supports continuous hydrogen production in air-grown microalgal cells.

Nitrogen availability modulates CO2 tolerance in a symbiotic chlorophyte

We studied acclimation to nitrogen starvation, a very high (20%) CO2 level, and combination thereof in a symbiotic chlorophyte Desmodesmus sp. 3Dp86E-1 to reveal potential mechanisms of its outstanding CO2 tolerance. The changes in growth rate, photosynthetic activity, cell pigment (chlorophyll and carotenoid), and elemental N and C contents were followed with special attention to rearrangement of the cell ultrastructure in the cultures grown with sparging by air or 20% CO2 in complete or N-free medium. Transcriptome of the microalga was screened for the presence of putative components of carbon concentrating mechanisms (CCM). The elevated CO2 was beneficial for the microalga whereas N-starvation impaired the buffer capacity, growth, and photosynthetic performance of the culture and promoted the accumulation of C-rich reserve compounds, degradation of the photosynthetic apparatus and pyrenoid. These effects were exacerbated under 20% CO2 in comparison with the air-grown cells. It is concluded that the exceptional CO2 tolerance in Desmodesmus sp. 3Dp86E-1 might have been acquired during co-evolution with the animal host. This trait is obviously conveyed to the symbiotic alga by concerted and swift responses of different mechanisms (CCM, photosynthetic apparatus, biosynthesis of reserve compounds) maintaining the pH homeostasis and carbon fixation and sink balance in the cell.

The impact of elevated CO2 concentration on the quality of algal starch as a potential biofuel feedstock

Cultured microalgae are viewed as important producers of lipids and polysaccharides, both of which are precursor molecules for the production of biofuels. This study addressed the impact of elevated carbon dioxide (CO2) on Chlorella sorokiniana production of starch and on several properties of the starch produced. The production of C. sorokiniana biomass, lipid and starch were enhanced when cultures were supplied with 2% CO2. Starch granules from algae grown in ambient air and 2% CO2 were analyzed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The granules from algae grown in 2% CO2 were disk-shaped and contained mainly stromal starch; granules from cultures grown in ambient air were cup-shaped with primarily pyrenoid starch. The granules from cells grown in 2% CO2 had a higher proportion of the accumulated starch as the highly branched, amylopectin glucan than did granules from cells grown in air. The rate of hydrolysis of starch from 2% CO2-grown cells was 1.25 times greater than that from air-grown cells and 2-11 times higher than the rates of hydrolysis of starches from cereal grains. These data indicate that culturing C. sorokiniana in elevated CO2 not only increases biomass yield but also improves the structure and composition of starch granules for use in biofuel generation. These modifications in culture conditions increase the hydrolysis efficiency of the starch hydrolysis, thus providing potentially important gains for biofuel production.

PHOTOSYNTHETIC INORGANIC CARBON ACQUISITION IN AN ACID-TOLERANT, FREE-LIVING SPECIES OF COCCOMYXA (CHLOROPHYTA)(1).

The processes of CO2 acquisition were characterized for the acid-tolerant, free-living chlorophyte alga, CPCC 508. rDNA data indicate an affiliation to the genus Coccomyxa, but distinct from other known members of the genus. The alga grows over a wide range of pH from 3.0 to 9.0. External carbonic anhydrase (CA) was detected in cells grown above pH 5, with the activity increasing marginally from pH 7 to 9, but most of the CA activity was internal. The capacity for HCO3 (-) uptake of cells treated with the CA inhibitor acetazolamide (AZA), was investigated by comparing the calculated rate of uncatalyzed CO2 formation with the rate of photosynthesis. Active bicarbonate transport occurred in cells grown in media above pH 7.0. Monitoring CO2 uptake and O2 evolution by membrane-inlet mass spectrometry demonstrated that air-grown cells reduced the CO2 concentration in the medium to an equilibrium concentration of 15 μM, but AZA-treated cells caused a drop in extracellular CO2 concentration to a compensation concentration of 27 μM at pH 8.0. CO2 -pulsing experiments with cells in the light indicated that the cells do not actively take up CO2 . An internal pool of unfixed inorganic carbon was not detected at the CO2 compensation concentration, probably because of the lack of active CO2 uptake, but was detectable at times before compensation point was reached. These results indicate that this free-living Coccomyxa possesses a CO2 -concentrating mechanism (CCM) due to an active bicarbonate-uptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.

Acclimation of wild‐type cells and CO2‐insensitive mutants of the green alga Chlorella ellipsoidea to elevated [CO2

CO(2)-insensitive mutants of the green alga Chlorella ellipsoidea were previously shown to be unable to repress an inorganic carbon-concentrating mechanism (CCM) when grown under 5% CO(2). When air-grown, wild-type (WT) cells were transferred to 5% CO(2), an abrupt drop of P(max) to 43% the original level of air-grown cells was observed within the initial 12 h. Photosynthetic affinities of WT cells to dissolved inorganic carbon (DIC) were maintained at high levels for the initial 4 d of acclimation, and then decreased gradually to lower levels over the next 6 d. In contrast to WT cells, the CO(2)-insensitive mutant, ENU16, exhibited a constant P(max) at maximum levels and a low K(1/2)[DIC] throughout the acclimation period. The rapid P(max) drop within 12 h of acclimation in WT cells was significantly reduced by treatment with 0.5 mm of 6-ethoxybenzothiazole-2-sulphonamide (EZA), a specific membrane-permeable inhibitor of carbonic anhydrase (CA), suggesting the participation of internal CAs in the temporary drop in P(max) in WT cells. WT and ENU16 cells were grown in controlled equilibrium [CO(2)], and the photosynthetic rate of each acclimated cell type was measured under equilibrated growth [DIC] conditions. In WT cells acclimated to 0.14-0.4% [CO(2)], K(1/2)[DIC] values increased as [CO(2)] increased, and the photosynthetic rates at growth DIC conditions were shown to decrease to about 70% the P(max) level in this intermediate [CO(2)] range. Such decreases in the net photosynthetic rates were not observed in ENU16. These results suggest that algal primary production could be depressed significantly under moderately enriched CO(2) conditions as a result of acquiring intermediate affinities for DIC because of their sensitive responses to changes in the ambient [CO(2)].

Inorganic carbon acquisition by the chrysophyte algaMallomonas papillosa

Photosynthetic characteristics of the chrysophyte alga Mallomonas papillosa Harris et Bradley were investigated to determine whether this species has some form of CO2-concentrating mechanism. The effect of external pH on the photosynthetic oxygen evolution rate of air-grown cells demonstrated an optimum in the pH range 5.0–7.0. This species lacked external carbonic anhydrase, and the cells had no capacity for direct bicarbonate uptake and had a low affinity for dissolved inorganic carbon. Measurement of the fluxes of CO2and O2in photosynthesizing cells at pH 7.0, using mass spectrometry, displayed no rapid uptake but only a slow depletion of CO2from the medium upon illumination. Furthermore, CO2uptake and O2evolution by M. papillosa was greatly reduced by iodoacetamide, an inhibitor of CO2fixation. The overall internal pH of M. papillosa was determined by distribution of14C-benzoic acid over the pH range 5.5–6.0 and [2-14C]-5,5-dimethyloxazolidine-2,4-dione over the pH range 6.5–7.0 between the cells and medium. As the external pH was lowered from 7.0 to 5.5, there was a decrease in the internal pH of M. papillosa cells from 8.31 to 7.75. The ΔpH was great enough to allow the intracellular accumulation of inorganic carbon by the diffusive uptake of CO2.Key words: bicarbonate uptake, chrysophyte, CO2uptake, internal pH, Mallomonas papillosa.

Inorganic carbon acquisition in two species of marine prymnesiophytes

Inorganic carbon acquisition has been investigated in the marine prymnesiophytes Dicrateria inornata and Ochrosphaera neapolitana. The presence of external and internal carbonic anhydrase (CA) was detected in air-grown cells of D. inornata by potentiometric assay and corroborated by mass spectrometry. Growth on 3% CO2 repressed external CA activity. Extracts of air-grown cells of O. neapolitana showed considerable internal CA activity, but no external CA activity was detected by mass spectrometry. Illumination of air-grown cells of D. inornata incubated with 100 μM H13CO3 and treated with the CA inhibitor acetazolamide caused a rapid drop in the extracellular CO2 concentration to a level below its equilibrium concentration indicating that cells were actively taking up CO2 from the medium. The rate of O2 evolution at the CO: compensation concentration was higher than could be supported by the uncatalysed formation of CO2 from HCO3, . demonstrating that the cells were taking up HCO3 −directly. Similar results were obtained with air-grown cells of O. neapolitana. Active CO2 transport in both species was not repressed by growth under high levels of CO2. The photosynthetic electron transport inhibitor 3-(3′ 4′-dichlorophenyl)-l, 1-dimethylurea (DCMU) inhibited CO2 uptake in air-grown cells of both species, demonstrating that the energy to support active CO2 transport was derived from photosynthetic linear electron transport. These results show that the physiological characteristics of the CO2 concentrating mechanism in related species do not appear to be correlated with the availability of CO2 in the surrounding medium.

Regulation of energy balance in photosystems in response to changes in CO2 concentrations and light intensities during growth in extremely-high-CO2-tolerant green microalgae.

Regulation of energy balance in photosystems in response to extremely-high-CO2 (40%) and low-CO2 (0.04%) stress was studied in extremely-high-CO2-tolerant green microalgae, Chlorococcum littorale and Chlorella sp. UK001. To investigate the energy input process, we assessed an F714/F685-ratio in a 77K fluorescence emission spectrum induced by 440-nm excitation in intact cells, which represents a ratio of fluorescence intensities derived from light-harvesting chlorophyll complexes in PSI and PSII. The F714/F685-ratio increased in several days after transferring C. littorale cells from air to 40% CO2, from 3% to 40% CO2 and from 3% to air. In all cases, the increase in the F714/F685-ratio was observed in high cell density culture, but no or a little increase was apparent in sparse cell density culture, when these cultures were illuminated at 250 micromol photon m-2 s-1. Even in the sparse culture, however, a similar increase in the F714/F685-ratio was observed when C. littorale cells were transferred from 3% to 40% CO2 at 20 micromol photon m-2 s-1. The cell density did not affect the F714/F685-ratio when CO2 concentration was kept at 3%. The activity of PSI electron (e-) transport was much higher in 40% CO2-grown cells than in 3% CO2-grown cells irrespective of the cell density during the culture, whereas the difference in PSII activity between them was small. The PSI activity at high cell density was higher also in air-grown cells than that in 3% CO2-grown cells. In both dense and sparse culture, 40% CO2-grown cells and air-grown cells showed higher relative quantum yield of PSI in the presence of DCMU than 3% CO2-grown cells, suggesting an increase in cyclic electron flow around PSI. Likewise, the increase in the F714/F685-ratio in response to the transfer to 40% CO2 was observed also in another extremely-high-CO2-tolerant alga, Chlorella sp. UK001. The possible role of the increases in the F714/F685-ratio, PSI/PSII activity ratio and cyclic e- transport activity in extremely-high-CO2 acclimation is discussed in comparison with low-CO2 acclimation.

Dissolved inorganic carbon concentration mechanism in Chlamydomonas moewusii

The dissolved inorganic carbon concentrating mechanism(s) of Chlamydomonas moewusii CC 55 was compared with C. reinhardtii strain 137. C. moewusii is similar to C. reinhardtii with respect to maximal rates of photosynthetic oxygen evolution, CO 2 fixation, respiration, and the ability to efficiently concentrate inorganic carbon. C. moewusii has a low, but measurable amount of external carbonic anhydrase (CA) that was not inhibited by acetazolamide (AZ), an inhibitor of periplasmic carbonic anhydrase (pCA) in C. reinhardtii. The K 0.5(CO 2) for air-grown C. moewusii is about 1 μM and the algal cells accumulated dissolved inorganic carbon (DIC) to a level of about 1 mM in 60 s. AZ did not inhibit CO 2 fixation and the DIC accumulation by air-grown cells of C. moewusii. The K 0.5(CO 2) for both species remains constant from pH 6.5 to 9.5 while K 0.5(HCO 3 -) increased logarithmically, which indicates that CO 2 is the apparent inorganic carbon species that enters the cells in both algae. Antiserum prepared against the 37 kDa peptide of pCA from C. reinhardtii was immunoreactive with polypeptides of 26, 28, and 32 kDa in C. moewusii. The periplasmic carbonic anhydrase (pCA) activity is a part of the dissolved inorganic carbon concentrating mechanism in C. reinhardtii, but C moewusii accomplished inorganic carbon accumulation without an AZ-sensitive pCA.

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO(2).

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO(2) concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1-4 days. In contrast, C. littorale cells which had been grown in 5% CO(2) (5% CO(2)-grown cells) could grow in 40% CO(2) without any lag period. When air-grown cells were transferred to 40% CO(2), the quantum efficiency of PS II (Phi(II)) decreased greatly, while no decrease in Phi(II) was apparent when the 5% CO(2)-grown cells were transferred to 40% CO(2). In contrast to air-grown cells, 5% CO(2)-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO(2)-grown cells to air, photosynthetic susceptibility to 40% CO(2) was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO(2). The intracellular pH value (pH(i)) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO(2) for 1-2 h, but no such decrease in pH(i) was apparent in the presence of EZA. Both air- and 5% CO(2)-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO(2) concentrations, grew in 40% CO(2) without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO(2). No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells.

Active transport of CO2 and bicarbonate is induced in response to external CO2 concentration in the green alga Chlorella kessleri

The time-course of induction of CO(2) and HCO(3)- transport has been investigated during the acclimation of high CO(2)-grown Chlorella kessleri cells to dissolved inorganic carbon (DIC)-limited conditions. The rate of photosynthesis of the cells in excess of the uncatalysed supply rate of CO(2) from HCO(3)- was taken as an indicator of HCO(3)- transport, while a stimulation of photosynthesis on the addition of bovine carbonic anhydrase was used as an indicator of CO(2) transport. The maximum rate of photosynthesis (Pmax) was similar for high CO(2)-grown and low CO(2)-grown cells, but the apparent whole cell affinity for DIC and CO(2) of high CO(2)-grown cells was found to be about 30-fold greater than in air-grown cells, which indicates a lower affinity for DIC and CO(2). It was found that HCO(3)- and CO(2) transport were induced in 5.5 h in cells acclimating to air in the light and in the presence and absence of 21% O(2), which indicates that a change in the CO(2)/O(2) ratio in the acclimating medium does not trigger induction of DIC transport. No active DIC transport was detected in high CO(2)-grown cells maintained on high CO(2) for 5.5 h in the presence of 5 mM aminooxyacetate, an aminotransferase inhibitor. These results indicate no involvement of photorespiration in triggering induction. Active DIC transport induction was inhibited in cells treated with 5 microgram ml(-1) cycloheximide, but was unaffected by chloramphenicol treatment, indicating that the induction process requires de novo cytoplasmic protein synthesis. The total DIC concentration eliciting the induction and repression of CO(2) and HCO(3)- transport was higher at pH 7.5 than at pH 6.6. The concentrations of external CO(2) required for the induction and repression of DIC transport were 0 and 120 microM, respectively, and was independent of the pH of the acclimation medium. Prolonged exposure to a critical external CO(2) concentration elicits the induction of DIC transport in C. kessleri.

Energy sources for HCO3- and CO2 transport in air-grown cells of synechococcus UTEX 625

Light-dependent inorganic C (Ci) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO2 or HCO3- uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular Ci pool of 20 mm, even though CO2 fixation was completely inhibited, indicating that cyclic electron flow was involved in the Ci-concentrating mechanism. When 200 m N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar Ci accumulation was observed, suggesting that linear electron flow could support the transport of Ci. When carbonic anhydrase was not present, initial CO2 uptake was greatly reduced and the extracellular [CO2] eventually increased to a level higher than equilibrium, strongly suggesting that CO2 transport was inhibited and that Ci accumulation was the result of active HCO3- transport. With 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-treated cells, Ci transport and accumulation were inhibited by inhibitors of CO2 transport, such as COS and Na2S, whereas Li+, an HCO3--transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, Ci transport and accumulation were not inhibited by COS and Na2S but were inhibited by Li+. These results suggest that CO2 transport is supported by cyclic electron transport and that HCO3- transport is supported by linear electron transport.

Simulation of C1fluxes in cyanobacteria: comparison between the carboxysome hypothesis and the equilibrium proposal

Inorganic carbon fluxes were simulated by a mathematical model using an equilibrium hypothesis for a wide range of conditions in a closed system composed of air-grown cells of Synechococcus UTEX 625 in a reaction vessel connected to a mass spectrometer. The metabolic scheme took into account the input fluxes of CO2and HCO3−transport into the cells, the output fluxes of CO2and HCO3−efflux, the diversion of Q toward the formation of the internal C2pool, and photosynthetic CO2fixation. The equations expressed the variation in concentration of each inorganic species outside and inside the cell as a function of time. The input fluxes were previously characterized by their kinetic constants (K1/2and Vm) both during initial uptake occurring upon illumination of the cells and under steady-state photosynthesis conditions. The efflux rates of the various Cispecies from the cells were investigated under a wide variety of experimental conditions. Using these efflux rates, the permeability coefficients of the cell for CO2and HCO3−were calculated previously. Using the kinetic constants for CO2and HCO3−transport, the permeability coefficients of the cell for CO2and HCO3−and the geometrical characteristics of the cells, the model simulated precisely the [HCO3−]/[CO2] ratio and the [CO2] and [O2] changes in the extracellular medium as well as the rate of filling of the internal Cipool under various conditions. Accurate fitting of experimental data with calculated values were possible only when the intracellular Cispecies were assumed to be in equilibrium throughout the entire cell volume. Results are discussed and compared with those given by previous hypotheses. Key words: Synechococcus UTEX 625, blue green algae, cyanobacteria, mathematical model, active CO2transport, active HCO3−transport, steady state, photosynthesis, Ciconcentrating mechanism.

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.

Measurement of the amount and isotopic composition of the CO2released from the cyanobacteriumSynechococcusUTEX 625 after rapid quenching of the active CO2transport system

Air-grown cells of the cyanobacterium Synechococcus UTEX 625 were suspended in a cuvette connected to a mass spectrometer and supplied with H13C18O3−to investigate the intracellular interconversion between CO2and HCO3−as determined from the isotopic composition of CO2appearing in the extracellular medium under a wide variety of experimental conditions. Upon injection of H13C18O3−to the cell suspension in the light, the extracellular [13C16O2] increased. As the CO2species were13C labelled, this demonstrated that the18O-depleted CO2was originating from the added H13C18O3−. A comparison of the rates of13C16O16O appearance in the medium with the formation of13C16O16O from spontaneous dehydration–hydration in the extracellular medium in the presence of cells demonstrated that most of it had to originate from a series of intracellular dehydration–hydration cycles of H13C18O3−that had been recently transported into the cells. During the time course of the experiments both the m/z (mass to charge) = 49 (i.e.,13C18O18O) and 47 (i.e.,13C18O16O) signals decreased constantly, whereas the m/z = 45 signal (i.e.,13C16O2) always increased. Inhibiting CO2fixation enhanced the amount of CO2arising in the medium but did not change its isotopic composition, and the CO2was always fully depleted of18O. When the CO2transport system was inhibited by darkening the cells, adding inhibitors such as Na2S or COS, or quenching the uptake of inorganic13C with an excess of inorganic12C, the magnitude of the extracellular [13C16O2] was increased but the CO2species were still always depleted of18O. Various incubation times of the illuminated cells in the presence of H13C18O3−were used to obtain a variety of internal Cipool sizes. When the inhibitor (COS) was added, the amount of13C16O2arising during the response time of the mass spectrometer was equivalent to the amount of CO2that would have been present in the whole cell if CO2and HCO3−were in equilibrium throughout the entire cell volume, but it was at least 40 times higher than the amount of CO2that would have been present in the cell if the CO2was confined to the carboxysomes. Experiments were also conducted at pH 9.0 where the spontaneous rate of13C16O2production from H13C1803−dehydration–hydration would be negligible, and again the same features were observed. Results show that intracellular HCO3−and CO2are in rapid equilibrium throughout the entire cell volume. Key words: Synechococcus UTEX 625, cyanobacteria, CO2leakage,18O exchange, active CO2transport, carboxysomes, inorganic C concentrating mechanism.

Effect of the intracellular inorganic carbon pool on chlorophyll a fluorescence quenching and O2photoreduction in air-grown cells of the cyanobacteriumSynechococcusUTEX 625

Simultaneous measurements were made of O2exchange, inorganic carbon (Ci) accumulation and assimilation, and chlorophyll a fluorescence of the cyanobacterium Synechococcus UTEX 625. The addition of Cito cells at the CO2compensation point resulted in quenching of chlorophyll a fluorescence in the presence or absence of the CO2fixation inhibitor, iodoacetamide. The magnitude of quenching was related to electron flow to terminal electron acceptors such as CO2and O2. When photosynthetic CO2fixation was allowed, the rate of electron transport, as expressed by (F*m– F)/F*m, was highly correlated with the onset of photosynthesis. When CO2fixation was inhibited by the addition of iodoacetamide, the observed fluorescence quenching was consistent with the enhanced rate of O2photoreduction that occurred when Ciwas added. There was a close correlation (r = 0.98) between the magnitude of O2-dependent fluorescence quenching and the amount of O2photoreduction. The degree of stimulation of electron flow to O2photoreduction was dependent on the inorganic carbon concentration. The K1/2(Ci) for extracellular Ciwas 1.36 ± 0.13 μM (mean ± SD, n = 3) and K1/2(Ci) for the intracellular Cipool was 1.4 ± 0.18 mM (mean ± SD, n = 3). The reduction of N,N-dimethyl-p-nitrosoaniline was also stimulated by the addition of Ci, whereas the addition of Cihad no effect on the reduction of 2,6-dimethylbenzoquinone and ferricyanide. The results suggest that Cistimulates electron flow in photosystem I. Key words: cyanobacteria, O2photoreduction, fluorescence, Ciconcentrating mechanism, inorganic carbon pool, linear electron transport, kinetic study.

Oxygen photoreduction and its effect on CO2accumulation and assimilation in air-grown cells ofSynechococcusUTEX 625

Mass spectrometric measurements of16O2,18O2, and13CO2were used to measure the rates of gross O2evolution, O2uptake, and CO2assimilation in relation to light intensity, temperature, pH, and O2concentration by air-grown cells of the cyanobacterium Synechococcus UTEX 625. CO2fixation and O2photoreduction increased with increased light intensity and, although CO2fixation was saturated at 250 μmol ∙ m−2 ∙ s−1, O2photoreduction was not saturated until about 550 μmol ∙ m−2 ∙ s−1. At high light intensity addition of inorganic carbon to the cells stimulated O2photoreduction 2-fold when CO2, fixation was allowed and 5-fold when CO2, fixation was inhibited with iodoacetamide. The ability of O2, to act as an acceptor of photosynthetically generated reducing power was dependent upon the O2concentration, and the substrate concentration required for half maximum rate (K½(O2)) was 53.2 ± 4.2 μM (mean ± SD, n = 3). The Q10for oxygen photoreduction was about 2. A certain amount (10%) of O2appeared to be required for maximum photosynthesis, as photosynthesis was inhibited under anaerobic conditions, especially at high light intensity. The point of inhibition is unknown but it seemed unlikely to be on CO2transport or the concentration of intracellular dissolved inorganic carbon (Ci), as the rate of initial CO2transport was enhanced and the intracellular Q1pool increased in size under anaerobic conditions. Key words: cyanobacteria, photosynthesis, Ciconcentrating mechanism, inorganic carbon pool, O2photoreduction, electron transport, temperature.

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.

Characterisation of carbon dioxide and bicarbonate transport during steady-state photosynthesis in the marine cyanobacterium Synechococcus strain PCC7002

Net O2 evolution, gross CO2 uptake and net HCOinf3su− uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO2 concentrations. Regardless of the CO2 concentration during growth, all cells had the capacity to transport both CO2 and HCOinf3su−; however, the activity of HCOinf3su− transport was more than twofold higher than CO2 transport even in cyanobacteria grown at high concentration of inorganic carbon (Ci = CO2 + HCOinf3su−). In low-Ci cells, the affinities of CO2 and HCOinf3su− transport for their substrates were about 5 (CO2 uptake) and 10 (HCOinf3su− uptake) times higher than in high-Ci cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated Ci pool reached 18, 32 and 55 mM in high-Ci, air-grown and low-Ci cells, respectively, when measured at 1 mM external Ci. Photosynthetic O2 evolution, maximal CO2 and HCOinf3su− transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO2 provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO2 concentrations. Transport studies with high-Ci cells revealed that CO2 and HCOinf3su− uptake were equally inhibited when CO2 fixation was reduced by the addition of glycolaldehyde. In contrast, in low-Ci cells steady-state CO2 transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO2 and HCOinf3su− uptake as well as O2 evolution in both cell types. In high-Ci cells, the degree of inhibition was similar for HCOinf3su− transport and O2 evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO2 was much more sensitive to the inhibitor than HCOinf3su− transport, with an apparent I50 value of around 250 μM ethoxyzolamide for CO2 uptake. The implications of our results are discussed with respect to Ci utilisation in the marine Synechococcus strain.

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.