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The eustigmatophyceans are primitive unicellular algae that represent the most basal group of ochrophytes. They are believed to be obligate photoautotrophs, occurring mainly in freshwater and soil but with some marine representatives. The freshwater eustigmatophytes Monodus subterraneus and Vischeria stellata, and the marine eustigmatophyte Nannochloropsis gaditana, have been studied by mass spectrometry with respect to their characteristics for inorganic carbon (Ci) uptake. A CO2 concentrating mechanism was found in all three, but an external carbonic anhydrase (CA) was not detected. The acquisition of Ci from the external medium was based on the active transport of HCO3–, CO2, or both. In particular, N. gaditana was able to use HCO3– exclusively as an exogenous carbon source for photosynthesis, with this HCO3– being subsequently converted to CO2 by an intracellular CA for photosynthetic fixation. A unique characteristic of this species was its capacity to transport HCO3– during prolonged periods of time in the dark. In contrast, M. subterraneus utilized CO2 alone through an active transport process, whereas V. stellataexhibited the capacity to transport both HCO3– and CO2. The uptake of CO2 also continued in the dark in V. stellatacells. Regardless of the Ci species taken up, transport was abolished by anoxia and by inhibitors of mitochondrial respiration. These results indicate that that the supply of Ci for photosynthetic CO2 fixation is partly dependent upon mitochondrial activity in these primitive eukaryotes.

Eukaryotic microalgae have developed CO2concentrating mechanisms to maximise the concentration of CO2 at the active site of Rubisco in response to the low CO2 concentrations in the external aquatic medium. In these organisms, the modes of inorganic carbon (Ci) uptake are diverse, ranging from diffusive CO2 uptake to the active transport of HCO3 -and CO2 and many have an external carbonic anhydrase to facilitate HCO3- use. There is unequivocal evidence for the mechanisms of Ci uptake in only about 25 species of microalgae of the chlorophyte, haptophyte, rhodophyte, diatom, and eustigmatophyte groups. Most of these species take up both CO2 and HCO3-, but the rates of uptake of each of these substrates varies with the algal species. A few species take up only one of the two forms of Ci, an adaptation that is not necessarily correlated with their ecological distribution. Evidence is presented for the active uptake of HCO3- and CO2 in two marine haptophytes,Isochrysis galbana Parke and Dicrateria inornata Parke, and for active transport of CO2 but lack of HCO3- uptake in two marine dinoflagellates, Amphidinium carteraeHulburt and Heterocapsa oceanica Stein.

The ability of the freshwater alga, Chlorella kessleri, to maintain a carbon concentrating mechanism when grown at acid pH was investigated. The alga grows over the pH range 4.0-9.0 and was found to take up bicarbonate and CO2 actively when grown at pH 6.0. However, when grown at acid pH (below 5.5), it does not have active CO2 uptake. The acidotolerant species maintained an internal pH of 6.1-7.5 over the external pH range 4.5-7.5, thus the pH difference between the cell interior and the external medium was large enough to allow for the diffusive uptake of CO2 at acid external pH. Mass spectrometric monitoring of O2 and CO2 fluxes by suspensions of C. kessleri, grown at acid pH, and maintained at pH 7.5 showed that the rates of O2 evolution did not exceed those of CO2 uptake. The final CO2 compensation concentrations of 14.0-17.7 µM reached by photosynthetic cells were above the CO2 equilibrium concentration in the external medium, indicating a lack of active CO2 uptake at acid pH. Chlorella kessleri accumulated CO2 with internal concentrations that were 9.9, 18.7 and 22.7-fold that of the external medium for cells grown, respectively, at pH 4.5, 5.0 and 5.5. The ability of C. kessleri cells to accumulate high intracellular concentrations of inorganic carbon at acid pH would provide a sufficiently high concentration of CO2 at the active site of Rubisco thus allowing the alga to maintain growth rates similar to those at alkaline pH.

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.

Acidification of the oceans, as a consequence of anthropogenic CO 2 emissions has reduced the pH at the surface of oceans by 0.1 unit compared to pre-industrial values. The reduction of seawater pH changes the relative proportion of the inorganic carbon (C i ) species which could potentially affect the modes of C i assimilation by marine microalgae. In this study the effects of changes in external pH on the modes of C i uptake over the pH range 5.0 to 7.5 were determined mass spectrometrically in Stichococcus minor Naegeli and S. cylindricus Butcher et Umbauk. Both species were found to tolerate a broad range of pH from pH 5.0 to 9.5 but the optimum for growth of both species was 8.2. Both species were also found to grow over a range of salinities and are best described as brackish water species rather than marine species, since they grow over wide ranges of salinity and pH. Neither species expresses external carbonic anhydrase (CA) activity. In both species, cells grown at pH 5.0, where the bulk of dissolved inorganic carbon (DIC) is in the form of CO 2 , active HCO 3 − and CO 2 uptake were absent and cells appear to take up CO 2 by diffusion. However, active HCO 3 − uptake was present in cells of both species grown at pH 6.0, 7.0 and 7.5 but active CO 2 uptake was not detectable. Cells of both species, when grown at pH 8.2, display both active CO 2 uptake and active HCO 3 − uptake.

The mechanism of inorganic carbon (Ci) uptake was examined in the marine green microalgae Stichococcus cylindricus and Stichococcus minor. External carbonic anhydrase (CA) activity was not detected in either species, by potentiometric assay or by mass spectrometry. Photosynthetic characteristics of Ci uptake indicate that both species have high apparent affinity for CO2 with a low K1/2(CO2) of about 10 µm. The O2 evolution rates in light exceeded the spontaneous CO2 formation rate by 2.5-fold in both species, which thus have active bicarbonate uptake. Mass spectrometric monitoring of CO2 and O2 fluxes showed that rates of O2 evolution exceeded those of CO2 depletion by about three- and twofold in S. minor and S. cylindricus, respectively, and also showed, in cells photosynthesizing at pH 8.2, a rapid depletion of CO2 upon illumination to a CO2 compensation concentration of 15.42 and 12.03 µm in S. minor and S. cylindricus, respectively. Both species also exhibit active CO2 uptake: addition of bovine CA at CO2 compensation concentration caused a rapid rise in CO2 as the CO2–HCO3- equilibrium was restored. Accumulation of unfixed Ci by cells at pH 8.2 was calculated to be 84.33 mm in S. cylindricus, and 30.37 mm in S. minor to give internal accumulations of 23- and 8-fold, respectively, compared to the external Ci concentration.

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 lM, but AZA-treated cells caused a drop in extracellular CO2 concentration to a compensation concentration of 27 l Ma t pH 8.0. CO 2pulsing experiments with cells in the light indicated that the cells do not actively take up CO2 .A n 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 bicarbonateuptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.

Germination and dormancy-breaking requirements have been investigated of Chara vulgaris , L. oospores harvested from surface sediments and from the plants of two lakes, Schumacher and Little Pearl in the locality of Timmins, northern Ontario. Oospores exhibited contrasting germination rates of 4–5% for fresh and 28–45% for sedimentary oospores. Dormancy of sedimentary oospores was readily broken by after-ripening for 60 days at 4 °C followed by exposure to white or red light, or by gibberellic acid (GA) in light (49–74% germination), whereas fresh oospores were less responsive (12–22% germination) to such treatments. Treatment of oospores by stratification or with GA or exposure to red light caused the induction of α-amylase activity. Abscisic acid reduced the effect of stratification and inhibited the GA-enhanced germination of sedimentary oospores, providing evidence of the participation of a phytochrome mechanism and hormonal regulation in the breaking of oospore dormancy. These results indicate that C. vulgaris oospores exhibit primary dormancy when first released from the parent plant, which is broken by exposure to low temperature, followed by secondary dormancy which is broken by a further period of stratification and exposure to light. Oospores from both lakes responded similarly to the dormancy-breaking treatments, but their overall viabilities were significantly different.

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)].

Carbonic anhydrase (CA) isozymes were identified and isolated from three strains of Phaeodactylum tricornutum [University of Texas Culture Collection (UTEX 640), North Eastern Pacific Culture Collection at the University of British Columbia B31 and Culture Collection of Algae and Protozoa 1052/1A]. External (CA ext ) and internal CA activity was detected by potentiometric assay of intact cells and cell homogenates of air and high CO 2 -grown cells. CA ext was detected only in UTEX 640 grown under CO 2 -limited conditions and present in trace amounts in cells grown on high CO 2 . CA isozymes in cells extracts were separated by cellulose acetate electrophoresis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All three strains had two CA bands in common, while UTEX 640 had a third, faster-running band which was absent from extracts of high CO 2 -grown cells and thus was the external isozyme. The internal CA isoforms of the UTEX 640 strain were shown to have molecular masses of 28 and 25 kDa, and the external 24 kDa. A fourth CA ext isozyme with a molecular weight of 23.5 kDa was later detected using a polyclonal CA antibody. The CA isozymes were low-CO 2 -inducible proteins because Western blot analysis, using a polyclonal antibody, indicated that CA expression was repressed in high CO 2 -grown cells. CA localization, using both immunofluorescence and immunogold techniques, with air-grown cells indicated that the CA ext was located in the periplasmic space and on the cell membrane, whereas in high CO 2 -grown cells only internal CA was detected.

The presence and characteristics of the CO2-concentrating mechanism in the freshwater eustigmatophyte Eustigmatos vischeri was investigated. The capacity of cells to remove CO2 actively from the medium was established by mass spectrometry; illumination of a cell suspension incubated with inorganic carbon (Ci) caused a rapid drop in the extracellular CO2 concentration to levels below its equilibrium value with bicarbonate. No activity of external carbonic anhydrase was detected in these experiments. The occurrence of the active uptake of CO2 was corroborated when cells were pulsed with pure CO2 in the light and in the dark and the rates of CO2 disappearance were faster than those measured in a buffer solution. CO2 transport in the light and in the dark was inhibited by low concentrations (100 μM) of the mitochondrial respiration inhibitor cyanide, and cells treated with azide, which effectively blocks mitochondrial complex IV, also lost their capacity to remove CO2. The ability of cells to take up bicarbon...

The mechanism of inorganic carbon uptake was examined in Euglena gracilis Klebs. and the acidophilic species Euglena mutabilis Schmitz. Both species, whether grown in acidic (pH 3.5) or alkaline (pH 7.5) media lack external carbonic anhydrase. Acid-grown E. gracilis was shown to have no capacity for bicarbonate transport, but transport was induced on transfer to alkaline medium (pH 7.5) in the light over a period of 8 h. In contrast, acid-grown E. mutabilis appears to have no capacity for bicarbonate transport even at neutral pH. The overall internal pH of the cells was determined by equilibration with14C-labelled benzoic acid over the pH range 3.5–5.0 and with14C-labelled 5,5-dimethyloxazolidine-2,4-dione over the range pH 5.5–7.5. The acidophilic species maintains an internal pH range of 6.6–6.8 in an external pH range of 3.5–5.5, whereas the acid-tolerant species E. gracilis maintains a neutral internal pH in an external pH range of 3.5–7.5. Measurement, by mass spectrometry, of the fluxes of CO2and O2in photosynthesizing cells at pH 3.5 demonstrated a rapid uptake of CO2by both species that was completely blocked by iodoacetamide, an inhibitor of CO2fixation. Uptake of CO2by E. gracilis, grown at pH 7.5, was not completely inhibited by iodoacetamide and O2evolution was sustained when the cells reached the CO2compensation concentration, indicating a direct uptake of bicarbonate. These data indicate that the acidophilic species, E. mutabilis, takes up CO2by diffusion, whereas the acid-tolerant species, E. gracilis, takes up CO2by diffusion at acid pH levels but has some capacity for active bicarbonate uptake when grown at alkaline pH levels.Key words: acidophilic alga, acidotolerant alga, bicarbonate uptake, CO2uptake, Euglena gracilis, Euglena mutabilis, internal pH.

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.

The effect of external pH and high CO2 were investigated on growth and photosynthesis in two marine dinoflagellates, Amphidinium carterae Hulburt and Heterocapsa oceanica Stein. Exposure of both species to high CO2 caused a rapid decrease in external pH from 8 to 7 and a complete suppression of growth. Both species were able to grow at pH 7 but the Vmax of photosynthesis was significantly lower than in cells grown at pH 8. There was also a significant reduction in the photosynthetic rate of both species after being on high CO2 for 6 h and a complete loss after 24 h. The internal pH of both species was determined by the distribution of [2-14C]5,5-dimethyloxazolidine-2,4-dione between the cells and the surrounding medium. As the external pH was lowered from 8 to 7, there was a decrease in the internal pH of A. carterae cells, grown at pH 8.0, from 7.92 to 7.04 and in H. oceanica from 8.14 to 7.22. When placed on high CO2, the internal pH of A. carterae further declined to 6.90 and to 7.15. in H. oceanica. This inability to maintain internal pH probably caused the suppression of growth and loss of photosynthetic capacity of high-CO2 cells.Key words: Amphidinium carterae, CO2-concentrating mechanism, dinoflagellates, external pH, Heterocapsa oceanica, internal pH.

Inorganic carbon uptake was investigated in two marine dinoflagellates, Amphidinium carterae Hulburt and Heterocapsa oceanica Stein. Mass spectrometric and potentiometric assays indicated that both species lacked external carbonic anhydrase (CA). The presence of internal CA was demonstrated by potentiometric assay and by the inhibition of photosynthesis upon the addition of 500lM ethoxyzolamide a membrane-permeable inhibitor of CA. The capacity for bicarbonate transport was investigated by comparing the calculated rate of spontaneous CO2 formation at pH 8.2 and 251 C with the rate of photosynthesis after the addition of 100lM NaHCO3. Both species appeared to have a very limited capacity for direct bicarbonate uptake. Monitoring of CO2 and O2 fluxes in both species by mass spectrometry demonstrated a rapid uptake of CO2 on illumination, to concentrations below the CO2 equilibrium concentration, indicating an effective selective uptake of CO2. This dependence of photosynthesis on free CO2alone suggests thatthese species are CO2 limited in their natural environment because the CO2 concentration of seawater is very low. Key index words: Amphidinium carterae; carbonic anhydrase; CO2-concentrating mechanism; CO2 uptake; dinoflagellates; Heterocapsa oceanica Abbreviations: CA, carbonic anhydrase; CCM, CO2concentrating mechanism; Ci, inorganic carbon; DIC, dissolved inorganic carbon; EZA, ethoxyzolamide

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 resul...

Inorganic carbon acquisition has been investigated in the marine haptophyte Isochrysis galbana. External carbonic anhydrase (CA) was present in air-grown (0.034% CO 2 ) cells but completely repressed in high (3%) CO 2 -grown cells. External CA was not inhibited by 1.0 mM acetazolamide. The capacity of cells to take up bicarbonate was examined by comparing the rate of photosynthetic O 2 evolution with the calculated rate of spontaneous CO 2 supply; at pH 8.2 the rates of O 2 evolution exceeded the CO 2 supply rate 14-fold, indicating that this alga was able to take up HCO 3 -. Monitoring CO 2 concentrations by mass spectrometry showed that suspensions of high CO 2 -grown cells caused a rapid drop in the extracellular CO 2 in the light and addition of bovine CA raised the CO 2 concentration by restoring the HCO 3 --CO 2 equilibrium, indicating that cells were maintaining the CO 2 in the medium below its equilibrium value during photosynthesis. A rapid increase in extracellular CO 2 concentration occurred on darkening the cells, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. Active CO 2 uptake was blocked by the photosynthetic electron transport inhibitor 3-(3',4'-dichlorphenyl)-1,1-dimethylurea, indicating that CO 2 transport was supported by photosynthetic reactions. These results demonstrate that this species has the capacity to take up HCO 3 - and CO 2 actively as sources of substrate for photosynthesis and that inorganic carbon transport is not repressed by growth on high CO 2 , although external CA expression is regulated by CO 2 concentration.

The CO2-concentrating mechanism (CCM) of the marine eustigmatophycean microalga Nannochloropsis gaditana consists of an active HCO3 −transport system and an internal carbonic anhydrase to facilitate accumulation and conversion of HCO3 − to CO2 for photosynthetic fixation. Aqueous inlet mass spectrometry revealed that a portion of the CO2 generated within the cells leaked to the medium, resulting in a significant rise in the extracellular CO2 concentration to a level above its chemical equilibrium that was diagnostic for active HCO3 − transport. The transient rise in extracellular CO2 occurred in the light and the dark and was resolved from concurrent respiratory CO2efflux using H13CO3 − stable isotope techniques. H13CO3 −pump-13CO2 leak activity of the CCM was unaffected by 10 μm3(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of chloroplast linear electron transport, although photosynthetic O2evolution was reduced by 90%. However, low concentrations of cyanide, azide, and rotenone along with anoxia significantly reduced or abolished 13CO2 efflux in the dark and light. These results indicate that H13CO3 − transport was supported by mitochondrial energy production in contrast to other algae and cyanobacteria in which it is supported by photosynthetic electron transport. This is the first report of a direct role for mitochondria in the energization and functioning of the CCM in a photosynthetic organism.

We examined the mechanism(s) responsible for active uptake of dissolved inorganic carbon (DIC) during photosynthesis in the green alga Eremosphaera viridis De Bary. O2 electrode measurements of algal oxygen evolution and CO2 fluxes as a function of DIC availability indicate that E. viridis actively imports only CO2 during photosynthesis, and does not possess external carbonic anhydrase (CA). The K0.5[CO2] was 14.2 and 10.1 µM at pH 5.0 and 8.0, respectively. Both membrane potential and cellular resistance were measured under controlled conditions of [CO2] at either 2 or 15 µM. Active CO2 uptake was electrically silent, suggesting that CO2 uptake might be mediated by a CO2-ATPase. Comparison of ATPase activity in microsomal preparations at low (0 µM) and high (15 µM) [CO2] indicated a 1.25-fold increase in ATP hydrolysis in high [CO2]. The CO2-ATPase activity was inhibited by the broad-acting inhibitors diethylstilbestrol (DES) and N',N'-dicyclohexylcarbodiimide (DCCD) but unaffected by vanadate, fluoride, and nitrate. The K0.5[CO2] of the ATPase activity was 22.5 µM, a value very similar to the K0.5[CO2] for CO2 uptake by whole algal cells. These results suggest the existence of a CO2-ATPase as the major importer of DIC for photosynthesis in the microalga E. viridis.Key words: chlorophyte, CO2 transport, CO2-ATPase, photosynthesis, electrical potential, mass spectrometry.

Physiological properties of photosynthesis were determined in the marine diatom, Phaeodactylum tricornutum UTEX640, during acclimation from 5% CO 2 to air and related to H 2 CO 3 dissociation kinetics and equilibria in artificial seawater. The concentration of dissolved inorganic carbon at half maximum rate of photosynthesis (K 0.5 [DIC]) value in high CO 2 -grown cells was 1009 mmol m -3 but was reduced three-fold by the addition of bovine carbonic anhydrase (CA), whereas in air-grown cells K 0.5 [DIC] was 71 mmol m -3 , irrespective of the presence of CA. The maximum rate of photosynthesis (P max ) values varied between 300 and 500 μmol O 2 mg Chl -1 h -1 regardless of growth pCO 2 . Bicarbonate dehydration kinetics in artificial seawater were re-examined to evaluate the direct HCO 3 - uptake as a substrate for photosynthesis. The uncatalysed CO 2 formation rate in artificial seawater of 31.65‰ of salinity at pH 8.2 and 25 °C was found to be 0-6 mmol m -3 min -1 at 100 mmol m -3 DIC, which is 53.5 and 7.3 times slower than the rates of photosynthesis exhibited in air- and high CO 2 -grown cells, respectively. These data indicate that even high CO 2 -grown cells of P. tricornutum can take up both CO 2 and HCO 3 - as substrates for photosynthesis and HCO 3 - use improves dramatically when the cells are grown in air. Detailed time courses were obtained of changes in affinity for DIC during the acclimation of high CO 2 -grown cells to air. The development of high-affinity photosynthesis started after a 2-5 h lag period, followed by a steady increase over the next 15 h. This acclimation time course is the slowest to be described so far. High CO 2 -grown cells were transferred to controlled DIC conditions, at which the concentrations of each DIC species could be defined, and were allowed to acclimate for more than 36 h. The K 0.5 [DIC] values in acclimated cells appeared to be correlated only with [CO 2(aq) ] in the medium but not to HCO 3 - , CO 3 2- , total [DIC] or the pH of the medium and indicate that the critical signal regulating the affinity of cells for DIC in the marine diatom, P. tricornutum, is [CO 2(aq) ] in the medium.

The effects of the carbonic anhydrase (CA) inhibitors acetazolamide (AZ) and dextran-bound sulfonamide (DBS) on HCO 3 - -dependent O 2 evolution in Chlorella saccharophila were evaluated. Addition of 4 μM AZ or 0.4 mg ml -1 DBS to photosynthesizing cells reduced the O 2 evolution rate at low dissolved inorganic carbon (DIC) concentration, decreased the size of the intracellular acid-labile carbon pool, and decreased the apparent affinity of the cells for DIC. Measurement of the whole-cell affinity of cells for CO 2 and HCO 3 - in the presence and absence of inhibitors indicated that active HCO 3 - transport was inhibited by AZ and DBS. The inhibition of HCO 3 - transport was independent of the inhibition of external and internal CA. These results suggest that the active uptake of HCO 3 - occurs initially by the interaction of HCO 3 - and a CA-like transporter.

Induction of the carbon concentrating mechanism (CCM) has been investigated during the acclimation of 5% CO 2 -grown Chlamydomonas reinhardtii 2137 mt + cells to well-defined dissolved inorganic carbon (C i ) limited conditions. The CCM components investigated were active HCO 3 - transport, active CO 2 transport and extracellular carbonic anhydrase (CA ext ) activity. The CA ext activity increased 10-fold within 6 h of acclimation to 0.035% CO 2 and there was a further slight increase over the next 18 h. The CA ext activity also increased substantially after an 8 h lag period during acclimation to air in darkness. Active CO 2 and HCO 3 - uptake by C. reinhardtii cells were induced within 2 h of acclimation to air, but active CO 2 transport was induced prior to active HCO 3 - transport. Similar results were obtained during acclimation to air in darkness. The critical C i concentrations effecting the induction of active C i transport and CA ext activity were determined by allowing cells to acclimate to various inflow CO 2 concentrations in the range 0.035-0.84% at constant pH. The total C i concentration eliciting the induction and repression of active C i transport was higher during acclimation at pH 7.5 than at pH 5.5, but the external CO 2 concentration was the same at both pHs of acclimation. The concentration of external CO 2 required for the full induction and repression of C; transport and CA ext activity were 10 and 100 μM, respectively. The induction of CA ext and active C i transport are not correlated temporally, but are regulated by the same critical CO 2 concentration in the medium.

As previously described, the absolute rate of photosynthesis due to a limited concentration of dissolved inorganic carbon at alkaline pH, where the rate of CO2 formation is strictly limited, plotted as a function of chlorophyll (Chl) concentration, will take the form of a rectangular hyperbola combined with a linear rate directly proportional to [Chl], which are, respectively, due to the contribution of CO2 and HCO3– to photosynthesis. This model represents that the mathematical asymptote of absolute rate of photosynthesis versus cell density is described by the whole-cell rate constant for HCO3– uptake and the maximum rate of CO2 formation in the extracellular space. This means that any trace modification of the CO2 formation rate outside the cell will alter the photosynthetic rate and should be detectable experimentally. In air-grown Chlorella ellipsoidea and C. kessleri and in high CO2-grown C. saccharophila, the graph of the absolute rate of photosynthesis against [Chl] clearly followed the mathematical model described above and the actual CO2 formation rates outside the cells were not significantly different from the calculated rates. It also indicated that the whole-cell rate constants for CO2 and HCO3– uptake in air-grown C. ellipsoidea and C. saccharophila were similar at ≈ 300 and 2·0 mm3μg–1 Chl min–1, respectively, whereas those in air-grown C. kessleri were ≈ 550 and 15 mm3μg–1 Chl min–1. These results indicate that no acidification of the periplasmic space occurs, and there is no trace activity of external carbonic anhydrase in these microalgae.

The regulation of the expression of the inorganic carbon concentrating mechanism (CCM) in aquatic photoautotrophs, particularly green algae, has been thought to require light and active photosynthesis. Recent studies, however, have indicated that there may be a light-independent pathway of signal transduction in green algae that may regulate the expression of CCMs in response to changes in ambient dissolved inorganic carbon (DIC) concentration. In the green alga, Chlorella ellipsoidea, changes in the rate of transport of both CO2 and HCO3- were shown to occur in response to the CO2 concentration in the bulk medium, independent of pH, whereas other inorganic carbon species, which might induce or repress DIC transport expression, were shown to change markedly with the pH of the medium. Furthermore, neither changes in the CO2 concentration around ribulose bisphosphate carboxylase-oxygenase (Rubisco) nor light were shown to be critical factors in regulating CCM expression in this alga. CO2-insensitive mutants of Chlorella ellipsoidea were recently isolated in which DIC transport operates constitutively at maximum activity. These results strongly suggest that a direct CO2 sensing mechanism may operate at the cell surface in Chlorella ellipsoidea and that this mechanism may trigger the repression of the expression of DIC transport in response to high CO2. Some supportive evidence for this hypothesis has also been obtained in other green algae, Chlamydomonas reinhardtii and Chlorella kessleri. The possibility of the occurrence of a direct sensing mechanism for CO2, its implications, and possible coexistence of other regulatory systems for CCM expression are discussed.Key words: green algae, Chlorella, signal transduction, CO2-insensitive mutants, CO2-sensing mechanism.

Electrophysiological measurements of the acidophilic alga Eremosphaera viridis De Bary explored the effects of low CO2 levels on both membrane potential and resistance. This procedure incorporates a double-barreled microelectrode and suction pipette system, coupled with an approximately CO2-free environment. A key requirement is an artificial pond water perfusion media that has been purged of dissolved inorganic carbon by being boiled and bubbled with nitrogen gas. Both membrane potential and resistance were measured at pH5 in both low-CO2 conditions (2µM) and high-CO2 conditions (14µM) in both light, where CO2 transport is known to be active, and dark, where CO2 transport is not active. To avoid dissolved inorganic carbon contamination of the perfusion media, a special chamber was constructed, featuring a laminar flow of nitrogen gas over the solution, which allowed for the manipulation of cells while preventing any contamination by CO2 from the air. Results indicate that the uptake of CO2 by the alga is electrically silent and, therefore, not the result of a symport or antiport cotransport system that would "drive" CO2 uptake by coupling it to the electrochemical gradient of ions such as protons or sodium. The uptake is most likely facilitated by a transporter directly coupled with ATP hydrolysis.Key words: Eremosphaera viridis, dissolved inorganic carbon, CO2-ATPase, electrophysiology.

Our objective was to investigate the sensitivity of growth to acid pH, and to compare acid-sensitivity of growth, photosynthesis, and respiration of copper-tolerant (B-4, X-Cu) and copper-intolerant (X-72) strains of Scenedesmus acutus f. alternans . Further, we wanted to assess the effects of pH on copper toxicity. We hypothesized that B-4 and X-Cu will be more acid tolerant than X-72, and that growth will be more acid-sensitive than photosynthesis and respiration. Optimal growth of the three strains occurred at circumneutral pHs where growth rates were similar. Growth rates responded differently to decreasing pHs. In B-4, the most acid-tolerant strain, growth was reduced by 50% at pH 4.42. X-Cu was less tolerant since it did not grow at pH 4.5, and showed 50% growth reduction at pH 4.81. X-72 was not acid-tolerant. Growth was reduced by 50% at pH 5.05, and no growth occurred at pH 4.8 or less. Photosynthesis was less inhibited by acid pHs than growth but the strain ranking was the same: B-4 was most acid-tolerant, followed by X-Cu and X-72. A reduction in respiration with declining pH was observed in X-72 and B-4 but not in X-Cu, where respiration was unaffected by pH. CO 2 compensation points for photosynthesis in all three strains showed a general decrease with pH of growth, and were lower in B-4 and X-Cu than in X-72. The inhibitory effect of copper on photosynthesis in X-72 and B-4 was reduced at lower pHs; copper toxicity in X-Cu was unaffected by pH. These findings suggest that algae such as X-Cu with a metal-exclusion mechanism can also exclude protons. In such algae, high H + concentration in the medium does not ameliorate copper toxicity. In contrast, in non-tolerant species (e.g. X-72) and in species that accumulate copper (e.g. B-4) high H + concentrations in the range suitable for growth ameliorate copper toxicity, possibly by competing with the metal for binding sites on the cell surface thereby decreasing copper uptake.

The pathway of serine synthesis was investigated in six species of cyanobacteria: Anabaena cylindrica, A. variabilis, A. flos-aquae, Coccochloris peniocystis, Gleocapsa alpicola and Phormidium molle. Activity of the enzymes phosphoglycerate (PGA) dehydrogenase (EC 1.1.1.95), phosphoserine transaminase (EC 2.6.1.52) and phosphoserine phosphatase (EC 3.1.3.3) was detected in vitro in all species, but no PGA phosphatase (EC 3.1.3.20) or hydroxypyruvate reductase (EC 1.1.1.26) activity could be detected. Metabolism of [1- 14 C]PGA by cell-free extracts of C. peniocystis resulted in the labelling of serine, alanine and aspartate, which represented 84, 11 and 4% of the labelled amino acid pool, respectively. Labelled serine isolated from these experiments was 100% carboxyl-labelled indicating that it was formed directly from PGA. The labelling of serine was markedly reduced by 5 mM phosphoserine. These in vitro findings indicate that cyanobacteria are capable of synthesizing serine directly from PGA, independent of ribulose-1,5-bisphosphate oxidation, and that the route of serine synthesis via phosphohydroxypyruvate and phosphoserine is the predominant pathway in these organisms.

The articles in this issue of the Canadian Journal of Botany arose from presentations given at The Fifth International Symposium on Inorganic Carbon Utilization by Aquatic Photosynthetic Organisms held from 24 to 28 August 2004 at Saint-Sauveur, Quebec, Canada. They represent the current state of our understanding of CO2-concentrating mechanisms in these organisms and highlight recent molecular, physiological, and ecological advances made in this field of study. The influence of CO2-concentrating mechanisms on global carbon sequestration, species diversity, and population dynamics are also explored.Key words: algae, CO2-concentrating mechanisms, cyanobacteria, photosynthesis.

A new method has been developed for screening algal photosynthetic mutants. This method uses autoradiography to assess gross photosynthetic 14C fixation by green algal colonies on agar plates and allows the identification of clones that differ in photosynthetic characteristics from wild-type cells. Three wild-type cells, high-CO2-grown Chlorella ellipsoidea, air-grown C. ellipsoidea, and air-grown Chlorella saccharophila, had K0.5 values for dissolved inorganic carbon (DIC) of 1083, 250, and 50 [mu]M, respectively, and as plaques on agar plates at Chl densities greater than 25 [mu]g cm-2 exhibited relative amounts of 14C fixation of 15, 55, and 100%, respectively. Cells of C. ellipsoidea were mutagenized with x-rays and screened by this method. Growth of C. ellipsoidea in high CO2 represses DIC transport and thus lowers its affinity for DIC. Five of the mutants detected by this method showed high-affinity photosynthesis similar to air-grown wild-type cells even when grown in high CO2. Seven other mutants when grown in high CO2 showed affinities for DIC intermediate between air-grown and high-CO2-grown wild-type cells. The affinities of high-CO2-grown mutants were reflected precisely in their capacities to accumulate DIC intracellularly. These results indicate that the mutants are fully or partially insensitive to the repressive effect of ambient CO2 concentration on DIC transport.

External carbonic anhydrase (CA) activity in Chlorella saccharophila is suppressed by growth at high dissolved inorganic carbon and at acid pH. External CA activity was shown to be suppressed by growth at pHs below 7.0, with total repression at pH5.0. Growth in the presence of the buffer 3-[N-Morpholino]propane-sulphonic acid (MOPS) between pH 7 and 8 suppressed CA activity. Cells grown at pH8.0 aerated at 6 dm3 h−1 exhibited external CA activity of 5 units mg−1 Chl once the dissolved inorganic carbon (DIC) was reduced to 300 mmol m−3, and this increased to 30 units mg−1 Chl over a period of 3d while the DIC dropped to 30mmol m−3. Cells aerated at 180 dm3 h−1 showed a similar trend in CA activity, although the onset was delayed by 1 d and the DIC did not drop below 300 mmol m−3. Cells grown at pH 7.8 near an air equilibrium DIC of 300 mmol m−3had no detectable external CA activity. It is probable that it is the CO2 supply to the cell, and not total DIC or HCO−3 which controls external CA activity. Cells grown at pH 5.0 had no detectable activity, although they reduced the CO2 concentration to 0.6 mmol m−3. The loss of CA upon transfer of air-grown cells to 10 mmol mol−1 CO2 took place over 48 h and was light dependent, while the loss upon transfer from alkaline pH to acid pH look place over 12 h and was independent of light. The effects of pH are independent of the response to CO2.

Regulation of transport of dissolved inorganic carbon (DIC) in response to CO 2 concentration in the external medium has been compared in two closely-related green algae, Chlorella ellipsoidea and Chlorella saccharophila. C. ellipsoidea, when grown in high CO 2 , had reduced activities of both CO 2 and HCO 3 - transport and DIC transport activities were increased after the cells had acclimated to air. However, high CO 2 -grown C. saccharophila had a comparable level of photosynthetic affinity for DIC to that of air-grown C. ellipsoidea and this was accompanied by a capacity to accumulate high internal concentrations of DIC. The high photosynthetic affinity and the high intracellular DIC accumulation did not change in cells grown in air except that the occurrence of external carbonic anhydrase (CA) in air-grown C. saccharophila stimulated the intracellular DIC accumulation in the absence of added CA. These data indicate that active DIC transport is constitutively expressed in C. saccharophila, presumably because this alga is insensitive to the repressive effect of high CO 2 on DIC transport. This strongly supports the existence of a direct sensing mechanism for external CO 2 in Chlorella species, but also indicates that external CA is regulated independently of DIC transport in Chlorella species.

Some physiological characteristics of photosynthetic inorganic carbon uptake have been examined in the marine diatoms Phaeodactylum tricornutum and Cyclotella sp. Both species demonstrated a high affinity for inorganic carbon in photosynthesis at pH7.5, having K 1/2 (CO 2 ) in the range 1.0 to 4.0 mmol m -3 and O 2- and temperature-insensitive CO 2 compensation concentrations in the range 10.8 to 17.6 cm 3 m -3 . Intracellular accumulation of inorganic carbon was found to occur in the light ; at an external pH of 7.5 the concentration in P. tricornutum was twice, and that in Cyclotella 3.5 times, the concentration in the suspending medium. Carbonic anhydrase (CA) was detected in intact Cyclotella cells but not in P. tricornutum, although internal CA was detected in both species. The rates of photosynthesis at pH 8.0 of P. tricornutum cells and Cyclotella cells treated with 0.1 mol m -3 acetazolamide, a CA inhibitor, were 1.5- to 5-fold the rate of CO 2 supply, indicating that both species have the capacity to take up HCO 3 - as a source of substrate for photosynthesis. No Na + dependence for HCO 3 - could be detected in either species. These results indicate that these two marine diatoms have the capacity to accumulate inorganic carbon in the light as a consequence, in part, of the active uptake of bicarbonate.

Mass spectromelry has been used to investigate the uptake of CO2 by two marine diatoms, Phaeodactylum tricornutum and Cyclotella sp. The time course of CO2 formation in the dark after addition of 100 mmol m−3 dissolved inorganic carbon (DIC) to cell suspensions showed that external carbonic anhydrase (CA) was not present in cells of P. tricornutum but was present in Cyclotella sp. In the absence of external CA, or when it was inhibited by 5 mmol m−3 acetazolamide, cells of both species preincubated with 100 mmol m−3 DIG rapidly depleted almost all of the free CO2 (3·2mmol m−31 at pH7·5) from the suspending medium within seconds of illumination and prior to the onset of steady-state photosynthesis. Addition of bovine CA quickly restored the HCO3−–CO2 equilibrium in the medium, indicating that the initial depletion of CO2 resulted from the selective uptake of CO2 rather than uptake of all DIG species. Transfer of cells to the dark caused a rapid increase in the CO2 concentration in the medium, largely as a result of the efflux of unfixed inorganic carbon from the cells. The measured CO2 uptake rates for both species accounted for 50% of the total DIG uptake at HCO3−–CO2 equilibrium, indicating that HCOHCO3− was also being taken up. These results indicate that both Phaeodactylum tricornutum and Cyclotella sp. have the capacity to transport CO2 actively against concentration and pH gradients.

Changes in the physiological properties of the green alga Chlorella ellipsoidea (UTEX 20) were determined during adaptation from high CO2 to air. Cells of C. ellipsoidea, grown in high CO2, had an extremely low affinity for dissolved inorganic carbon (DIC). However, high-affinity DIC transport was induced rapidly after switching to air, which caused a massive decrease in the DIC concentration in the medium. Rates of O2 evolution without added carbonic anhydrase (CA) were compared with calculated rates of uncatalyzed CO2 formation in the medium as a measure of active HCO3-uptake. Cells were found to be able to use HCO3- after 5 h of adaptation and this capacity increased during the next 17 h. The stimulation of O2 evolution upon CA addition was used as a measurement of active CO2 transport: such stimulation occurred 2 h after transfer and increased during the next 5 h. Increases in O2 evolution rates were correlated closely with an increasing capacity to accumulate intracellular pools of acid-labile DIC and with decreases in K1/2(CO2) and CO2-compensation point of the cells. Treatment of cells with cycloheximide (5 [mu]g mL-1) during adaptation completely inhibited DIC transport induction, whereas treatment with chloramphenicol (400 [mu]g mL-1) had no effect, indicating the requirement for cytoplasmic protein synthesis in the induction. These results suggest that both CO2 and HCO3- transport are induced upon transfer of cells from high CO2 to air and that there is a temporal separation between the induction of the two systems.

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

A method is described for the rapid separation of carbonic anhydrase (CA) isozymes by cellulose acetate membrane electrophoresis in which CA activity is detected using the pH-indicating dye, bromcresol purple. This method can detect bovine erythrocyte CA in a 0.3 mm 3 sample applied at a concentration of 100 ng cm -3 (total of 30 pg applied) while at higher concentrations three isozymes were observed. It was found, using a potentiometric technique, that intact cells of Anabaena flos-aquae (Cyanophyceae) and Chlorella ellipsoidea had no detectable activity while C. saccharophila and Chlamydomonas reinhardtii (Chlorophyceae) had external CA activity

The mechanism of inorganic carbon (C(i)) uptake was examined in the marine green microalgae Stichococcus cylindricus and Stichococcus minor. External carbonic anhydrase (CA) activity was not detected in either species, by potentiometric assay or by mass spectrometry. Photosynthetic characteristics of C(i) uptake indicate that both species have high apparent affinity for CO(2) with a low K(1/2) (CO(2)) of about 10 µm. The O(2) evolution rates in light exceeded the spontaneous CO(2) formation rate by 2.5-fold in both species, which thus have active bicarbonate uptake. Mass spectrometric monitoring of CO(2) and O(2) fluxes showed that rates of O(2) evolution exceeded those of CO(2) depletion by about three- and twofold in S. minor and S. cylindricus, respectively, and also showed, in cells photosynthesizing at pH 8.2, a rapid depletion of CO(2) upon illumination to a CO(2) compensation concentration of 15.42 and 12.03 µm in S. minor and S. cylindricus, respectively. Both species also exhibit active CO(2) uptake: addition of bovine CA at CO(2) compensation concentration caused a rapid rise in CO(2) as the CO(2) -HCO(3) (-) equilibrium was restored. Accumulation of unfixed C(i) by cells at pH 8.2 was calculated to be 84.33 mm in S. cylindricus, and 30.37 mm in S. minor to give internal accumulations of 23- and 8-fold, respectively, compared to the external C(i) concentration.

External carbonic anhydrase (CA) was detected in whole cells of alkaline-grown Chlorella saccharophila but was suppressed by growth at acid pH or growth on elevated levels of CO2. Internal CA activity was measured potentiometrically as an increase in activity in cell extracts over that of intact cells. Cells grown under all conditions had equal levels of internal CA activity. Two isozymes were identified after electrophoretic separation of soluble proteins on cellulose acetate plates. The fast isozyme was found in cells grown under all conditions, whereas the slow isozyme was found only in cells grown at alkaline pH. Western blot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antibodies produced against the periplasmic form of CA from Chlamydomonas reinhardtii revealed a single band at 39 kD, which did not change in intensity between growth conditions and was associated only with proteins eluted from the fast band. The slow isozyme was inactivated by incubation of cell extract at 30[deg]C and by incubation in 10 mM dithiothreitol, whereas the internal form was unaffected. These results indicate that external and internal forms of CA differ in structure and their activities respond differently to environmental conditions.

The fluxes of CO 2 and oxygen during photosynthesis by cell suspensions of Tessellaria volvocina and Mallomonas papillosa were monitored mass spectrometrically. There was no rapid uptake of CO 2, only a slow drawdown to compensation concentrations of 26 lM for T. volvocina and 18 lM for M. papillosa, when O 2 evolution ceased, indicating a lack of active bicarbonate uptake by the cells. Darkening of the cells after a period of photosynthesis did not cause rapid release of CO 2, indicating the absence of an intracellular inorganic carbon pool. However, upon darkening a brief burst of CO 2 was observed similar to the post-illumination burst characteristic of C3 higher plants. Treatment of the cells of both species with the membrane-permeable carbonic anhydrase inhibitor ethoxyzolamide had no adverse effect on photosynthetic rate, but stimulated the dark CO 2 burst indicating the dark oxidation of a compound formed in the light. In the absence of any active accumulation of inorganic carbon photosynthesis in these species should be inhibited by O 2. This was investigated in four synurophyte species T. volvocina, M. papillosa, Synura petersenii, and Synura uvella: Photosynthetic O 2 evolution rates in all four algae, measured by O 2 electrode, were significantly higher (40-50%) in media at low O 2 (4%) than in airequilibrated (21% O 2) media, indicating an O 2 inhibition of photosynthesis (Warburg effect) and thus the occurrence of photorespiration in these species. © Springer Science+Business Media B.V. 2011.

The formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis was investigated and the activities of enzymes of glycolate metabolism assayed. Photosynthetic 14CO2 incorporation was O2 insensitive and no labelled glycolate could be detected in cells incubated at 2 and 21% O2. Under conditions of 100% O2 glycolate comprised less than 1% of the acid-stable products indicating ribulose 1,5 bisphosphate (RuBP) oxidation only occurs under conditions of extreme O2 stress. Metabolism of [1-14C] glycolate indicated that as much as 62% of 14C metabolized was released as 14CO2 in the dark. Metabolism of labelled glycolate, particularly incorporation of 14C into glycine, was inhibited by the amino-transferase inhibitor amino-oxyacetate. Metabolism of [2-14C] glycine was not inhibited by the serine hydroxymethyltransferase inhibitor isonicotinic acid hydrazide and little or no labelled serine was detected as a result of 14C-glycolate metabolism. These findings indicate that a significant amount of metabolized glycolate is totally oxidized to CO2 via formate. The remainder is converted to glycine or metabolized via a glyoxylate cycle. The conversion of glycine to serine contributes little to glycolate metabolism and the absence of hydroxypyruvate reductase confirms that the glycolate pathway is incomplete in this cyanobacterium.

Mass spectrometry has been used to investigate the transport of COz in the freshwater diatom Navicula pelliculosa. The time course of C02 formation in the dark after addition of 100 mmol m~3 dissolved inorganic carbon (DIC) to cell suspensions showed that no external carbonic anhydrase (CA) was present in these cells. Upon illumination, cells pre-incubated at pH 7-5 with 100 mmol m~3 DIC, removed almost all free C02 from the medium at an initial rate of 28 5 /i?mol C02 mg~1 Chi h-1. Equilibrium between HCO3 and C02 in the medium occurred rapidly upon addition of bovine CA, showing that C02 depletion resulted from a selective uptake of C02 rather than an uptake of all inorganic carbon species. However, photosynthetic 02 evolution rate remained constant after C02 had been depleted from the medium indicating that photosynthesis is sustained primarily by active HCO3 uptake. Treatment of cells with 2-iodoacetamide (8-3 mol m-3) completely inhibited C02 fixation but had little effect on C02 transport since initial rates of C02 depletion were about 81% that of untreated cells. Transfer of iodoacetamide-treated cells to the dark caused a rapid increase in the C02 concentration in the medium largely due to the efflux of the unfixed intracellular DIC pool which was found to be about 194 times the concentration of that in the external medium. These results indicate that Navicula pelliculosa actively takes up molecular C02 against a concentration gradient by a process distinct from HCOJ transport.

A malate dehydrogenase (MDH) was characterized from the cyanobacterium Coccochloris peniocystis. The enzyme was purified approximately 180-fold and had a molecular weight of about 90000. The enzyme had a pH optimum of pH 6.7 to 7.5; a Km (malate) of 5.6 mM and Kms for NAD and NADP of 24 μM and 178 μM, respectively, although similar Vmax were obtained with either pyridine nucleotide. Enzyme activity was inhibited by ATP, citrate, oxalacetate, acetyl CoA and CoA. Enzyme assays with uniformly 14C-labelled malate caused no 14CO2 release, indicating this MDH is not a malic enzyme. Electrophoresis and S-200 gel filtration of the partially purified enzyme indicated a single MDH was present in this preparation. A second, less abundant, MDH was present in crude extracts. The presence of MDH in this organism is consistent with the operation of a glyoxylate cycle which, in the absence of a TCA cycle, would provide organic acids required in secondary carbon metabolism. ATP inhibition of MDH may allow for light regulation of MDH activity since, in the light, oxaloacetic acid is generated by phosphoenolpyruvate carboxylase activity.

The uptake and accumulation of inorganic carbon has been investigated in Chlorella ellipsoidea cells grown at acid or alkaline pH. Carbonic anhydrase (CA) was detected in ceil extracts but not in intact cells and CA activity in acid-grown cells was considerably less than that in alkali-grown cells. Both cell types demonstrates low K1/2 (CO2) values in the range pH 7.0–8.0 and these were unaffected by O2 concentration. The CO2 compensation concentrations of acid- and alkali-grown cells suspended in aqueous media were not significantly different in the range of pH 6.0–8.0, but at pH 5.0, the CO2 compensation concentrations of acid-grown cells (57.4cm3 m−3) were lower than those of alkali-grown cells (79.2cm3 m−3). The rate of photo-synthetic O2 evolution in the range pH 7.5–8.0 exceeded the calculated rate of CO2 supply two- to three-fold, in both acid- and alkali-grown cells, indicating that HCO3− was taken up by the cells. Accumulation of inorganic carbon was measured at pH 7.5 by silicone-oil centri-fugation, and the concentration of unfixed inorganic carbon was found to be 5.1 mol m−3 in acid-grown and 6.4mol m−3 in alkali-grown cells. These concentrations were 4.6- and 5.9-fold greater than in the external medium. These results indicate that photorespiration is suppressed in both acid- and alkali-grown cells by an intracellular accumulation of inorganic carbon due, in part, to an active uptake of bicarbonate.

A study has been made of the capacity of whole cells and intact, photosynthetically active, isolated chloroplasts of Chlorella ellipsoidea (UTEX 20) to take up CO2 by active transport. Assays for carbonic anhydrase activity and the monitoring of CO2 uptake by cells and chloroplasts was carried out by mass spectrometry. No external carbonic anhydrase was detected in whole cells or on the outer surface of the isolated chloroplasts. Upon illumination, whole cells rapidly depleted CO2 from the medium to a level below the equilibrium CO2 concentration before maximum photosynthetic O2 evolution rates were established. Addition of bovine carbonic anhydrase resulted in the reestablishment of equilibrium CO2 concentrations, indicating that the cells were actively and selectively depleting the medium of CO2. This CO2 uptake was inhibited by 10 μM diethylstilbestrol. No such rapid depletion of CO2 was observed with isolated intact chloroplasts, although the organelles demonstrated rates of photosynthetic O2 evolution of about 60% of the parent cells. Photosynthetic rates of chloroplast suspensions exceeded the rate of CO2 supply only twofold, indicating that chloroplasts have a limited ability for HCO3− uptake. Intact cells, however, use HCO3− readily. These results indicate that the principal location of inorganic carbon transporters, both for CO2 and HCO3−, is at the plasma membrane in this alga. Key words: Chlorella ellipsoidea, CO2 transport, bicarbonate transport, chloroplasts, mass spectrometry, carbonic anhydrase.

Mass spectrometry has been used to measure the rates of CO2 uptake of acid- and alkali-grown cells of the green algae Chlorella ellipsoidea (UTEX 20) and C. saccharophila (UTEX 27). The time course of CO2 formation on addition of 100mmol m−3 K2CO3 to cells in the dark was used as an assay for external carbonic anhydrase (CA). No external CA was detected in acid-grown cells of either species or in alkali-grown cells of C. ellipsoidea but was present in alkali-grown C. saccharophila. In the absence of external CA, or when it was inhibited by 5mmol m−3 acetazolamide, cells of both species, on illumination, rapidly depleted the free CO2 in the medium at pH 7.5 to near zero concentrations before maximum photosynthetic O2 evolution rates were established. Addition of bovine CA rapidly restored the equilibrium CO2 concentration in the medium, indicating that the cells were selectively taking up CO2. Transfer of cells to the dark caused a rapid increase in the CO2 concentration in the medium largely due to the efflux of inorganic carbon from the cells as CO2. This rapid light-dependent CO2 uptake takes place against pH and concentration gradients and, thus, has the characteristics of active transport.

Carbonic anhydrase (CA) activity and localization have been examined in two species of the eukaryotic green alga Chlorella. Mass spectrometric and potentiometric assays of CA activity indicate that C. ellipsoidea contains very little extracellular CA activity whereas C. saccharophila exhibits significant extracellular activity when grown at alkaline pH values. Extracellular CA activity appears to be correlated with the presence of a 36 kilodalton polypeptide that was detected immunologically using a polyclonal antibody directed against the 37 kilodalton Chlamydomonas CA monomer. Both Chlorella species and enzymatically isolated C. ellipsoidea chloroplasts also contain an immunologically similar 38 kilodalton polypeptide that may be a cytosolic or chloroplastic form of CA.

Some characteristics of photosynthesis of three synurophyte algae, Synura petersenii, Synura uvella and Tessellaria volvocina were investigated to determine the mechanism of inorganic carbon (C(i)) uptake. All three species were found to have no external carbonic anhydrase, no capacity for direct bicarbonate uptake and a low whole-cell affinity for C(i). The internal pH of S. petersenii determined using (14)C-benzoic acid and [2-(14)C]-5,5-dimethyloxazolidine-2,4-dione was pH 7.0-7.5, over an external pH range of 5.0-7.5. Thus, the pH difference between the cell interior of S. petersenii and the external medium was large enough, over the alga's growth range, to allow the accumulation of C(i) by the diffusive uptake of CO(2). Monitoring O(2) evolution and CO(2) uptake by suspensions of S. petersenii at pH 7.0 by mass spectrometry did not indicate a rapid uptake of CO(2), and the final CO(2) compensation concentration reached was 24 +/- 0.7 microM. Furthermore, when the cells were darkened, a brief burst of CO(2) occurred before a steady rate of dark respiration was established, suggesting a loss of CO(2) by photorespiration. An examination of the kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in homogenates of cells of S. petersenii, S. uvella and Mallomonas papillosa showed that values of the K(m) (CO(2)) were 28.4, 41.8 and 18.2 microM, respectively. These species lack the characteristics of cells with a CO(2)-concentrating mechanism because the cell affinity for C(i) appears to be determined by the relatively high CO(2) affinity of the Rubisco of these algae.