Influence of Varying CO(2) and Orthophosphate Concentrations on Rates of Photosynthesis, and Synthesis of Glycolate and Dihydroxyacetone Phosphate by Wheat Chloroplasts

Intact chloroplasts of wheat (Triticum aestivum) were isolated from mesophyll protoplasts. With decreasing concentrations of bicarbonate from 10 to 0.3 millimolar (pH 8.0), the optimal concentration of orthophosphate (Pi) for photosynthetic O₂ evolution decreased from a value of 0.1 to 0.2 millimolar to 0 to 0.025 millimolar. The extremely low Pi optimum for photosynthesis at the low bicarbonate levels of 0.3 millimolar was increased by lowering the O₂ concentration from 253 (21% gas phase) to 72 micromolar (6% gas phase). The relative amount of glycolate and dihydroxyacetone phosphate (DHAP) synthesized under high and low levels of bicarbonate and varying levels of Pi was determined. At low levels of bicarbonate, glycolate was the main product, whereas at high bicarbonate levels, DHAP was the main product. Most of the DHAP and glycolate was found in the extrachloroplastic fraction.     

UPTAKE,EFFLUX, AND PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON BY THE MARINE EUSTIGMATOPHYTE NANNOCHLOROPSIS SP.1

Uptake, efflux and utilization of inorganic carbon were investigated in the marine eustigmatophyte Nannochloropsis sp. grown under an air level of CO₂. Maximal photosynthetic rate was hardly affected by raising the pH porn 5.0 to 9.0. The apparent photosynthetic affinity for dissolved inorganic carbon (DIC) was 35 μM DIC between pH 6.5 to 9.0, but increased approximately threefold at pH 5.0 suggesting that HCO₃- was the main DIC species used from the medium. No external carbonic anhydrase (CA) activity could be detected by the pH drift method. However, application of ethoxyzolamide (an inhibitor of CA) resulted an a significant inhibition of photosynthetic O₂ evolution and carbon utilization, suggesting involvement of internal CA or CA-like activity in DIC utilization. Under high light conditions, the rate of HCO₃^? uptake and its internal conversion to CO₂ apparently exceeded the rate of carbon fixation, resulting in a large leak of CO₂ from the cells to the external medium. When the cells were exposed to low DIC concentrations, the ratio of internal to external DIC concentration was about eight. On the other hand, in the presence of 2 mM DIC, conditions prevailing in the marine environment, the internal concentration of DIC was only 50% higher than the external one.     

Oxygen inhibition of dissolved inorganic carbon uptake in unicellular green algae

Unicellular green algae have a dissolved inorganic carbon (DIC) concentrating mechanism, commonly known as the DIC pump, to concentrate inorganic carbon into cells and chloroplasts. The DIC pump activity is normally measured as the K_0.5(DIC) that equals the CO₂ plus HCO₃‐ concentration at a cited pH at which the rate of DIC‐dependent photosynthetic O₂ evolution is half‐maximal, or by the amount of intra‐cellular DIC accumulation in 15–60 s, using a limited amount of NaH¹⁴CO₃, measured by the silicone oil cen‐trifugation technique. The dissolved oxygen in the assay inhibits or reduces the DIC uptake by the cells of unicellular green algae Chlamydomonas reinhardtii Dangeard, strain 137 and in a cell wall‐less marine algae Dunaliella tertiolecta Butcher. The algal cells concentrated the highest amount of DIC when little or no oxygen was present in the assay medium. The results suggest that the amount of O₂ and DIC must be carefully monitored before DIC‐pump assay.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Glycolate Excretion and the Oxygen to Carbon Dioxide Net Exchange Ratio during Photosynthesis in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells were grown in high (5% v/v) or low (0.03% v/v) CO₂ concentration in air. O₂ evolution, HCO₃⁻ assimilation, and glycolate excretion were measured in response to O₂ and CO₂ concentration. Both low- and high-CO₂-grown cells excrete glycolate. In low-CO₂-grown cells, however, glycolate excretion is observed only at much lower CO₂ concentrations in the medium, as compared with high-CO₂-adapted cells. It is postulated that the activity of the CO₂-concentrating mechanism in low-CO₂-grown cells is responsible for the different dependence of glycolate excretion on external CO₂ concentration in low- versus high-CO₂-adapted cells.     

Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

Formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis

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.Abbreviations AAN aminoacetonitrile - AOA aminooxyacetate - DIC dissolved inorganic carbon - INH isonicotinic acid hydrazide - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PG phosphoglycolate - PGA phosphoglyceric acid - PGPase phosphoglycolate phosphatase - PR photorespiration - Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase - TCA trichloroacetic acid - RuBP ribulose-1,5-bisphosphate     

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO₂ requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO₂ concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O₂. In contrast to a CO₂ compensation concentration of near zero in wild type at all O₂ concentrations examined, the mutant exhibited a high, O₂-stimulated CO₂ compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of ¹⁴CO₂. At air levels of CO₂ and O₂, the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO₂ concentration. At ambient CO₂, wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO₂ limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO₂ concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO₂, creating a high internal CO₂ concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO₂ concentration.     

Photorespiration and Carbon Limitation Determine Productivity in Temperate Seagrasses

The gross primary productivity of two seagrasses, Zostera marina and Ruppia maritima, and one green macroalga, Ulva intestinalis, was assessed in laboratory and field experiments to determine whether the photorespiratory pathway operates at a substantial level in these macrophytes and to what extent it is enhanced by naturally occurring shifts in dissolved inorganic carbon (DIC) and O₂ in dense vegetation. To achieve these conditions in laboratory experiments, seawater was incubated with U. intestinalis in light to obtain a range of higher pH and O₂ levels and lower DIC levels. Gross photosynthetic O₂ evolution was then measured in this pretreated seawater (pH, 7.8–9.8; high to low DIC:O₂ ratio) at both natural and low O₂ concentrations (adjusted by N₂ bubbling). The presence of photorespiration was indicated by a lower gross O₂ evolution rate under natural O₂ conditions than when O₂ was reduced. In all three macrophytes, gross photosynthetic rates were negatively affected by higher pH and lower DIC. However, while both seagrasses exhibited significant photorespiratory activity at increasing pH values, the macroalga U. intestinalis exhibited no such activity. Rates of seagrass photosynthesis were then assessed in seawater collected from the natural habitats (i.e., shallow bays characterized by high macrophyte cover and by low DIC and high pH during daytime) and compared with open baymouth water conditions (where seawater DIC is in equilibrium with air, normal DIC, and pH). The gross photosynthetic rates of both seagrasses were significantly higher when incubated in the baymouth water, indicating that these grasses can be significantly carbon limited in shallow bays. Photorespiration was also detected in both seagrasses under shallow bay water conditions. Our findings indicate that natural carbon limitations caused by high community photosynthesis can enhance photorespiration and cause a significant decline in seagrass primary production in shallow waters.     

Model for the Relationships betweenCO2-Concentrating Mechanism, CO2 Fixation, and Glycolate Synthesis during Photosynthesis in Chlamydomonas reinhardtii

We constructed a mathematical model for simulating the relationshipsof extracellular concentration of dissolved inorganic carbon(DIC), the rates of photosynthetic CO₂ fixation and glycolatesynthesis, and the concentrations of intrachloroplast CO₂ andO₂ in Chlamydomonas reinhardtii. When we compared the photosyntheticrates of I0W-CO₂ (air)-grown C. reinhardtii measured experimentallyand the rates simulated with the incubation conditions in themodel, the model was found to function well. The calculatedrates for glycolate synthesis also matched the measured ratesbetween 80 to 200 µM extracellular DIC, found in the presenceof 1 mM aminooxyacetate. The conformity of the calculated ratesto the measured ones of the glycolate synthesis encouraged usto estimate the O₂ concentration at the active site of ribulosebisphosphate carboxylase/oxygenase; the results were 0.36 and0.40 mM at 80 and 200 µM extracellular DIC, respectively.These high concentrations of O₂ were due to stimulation of photosyntheticCO₂ fixation and further O₂ evolution by a CO₂- concentratingmechanism in the low-CO₂-grown cells. These cells were calculatedto consume 43% of ATP formed photosynthetically for CO₂ concentrationat 200 µM extracellular DIC. The model modified to simulatethese relationships in high-CO₂ (3 to 5% CO₂)-grown C. reinhardtiipredicted O₂ concentration in chloroplasts to be 0.36 mM ina 1% CO₂ atmosphere. This high concentration of O₂ caused activeglycolate synthesis at the measured rate in the high-CO₂-growncells even in the presence of 1% CO₂. The comparisons of themeasured and simulated rates of photosynthesis in low- and high-CO₂-grownC. reinhardtii indicated that no matter how the CO₂ accumulatedin the chloroplasts, it increased the O₂ concentration in theorganelles, and consequently enhanced glycolate synthesis. ¹This paper is the twenty-first in a series on glycolate metabolismin Euglena gracilis. (Received March 11, 1987; Accepted August 17, 1987)     

The role of external carbonic anhydrase in the photosynthetic use of inorganic carbon in the deep-water alga Phyllariopsis purpurascens (Laminariales, Phaeophyta)

Mechanisms of inorganic carbon assimilation were investigated in the deep-water alga Phyllariopsis purpurascens (C. Agardh) Henry et South (Laminariales, Phaeophyta). The gross photosynthetic rate as a function of external pH, at a constant concentration of 2 mM dissolved inorganic carbon (DIC), decreased sharply from pH 7.0 to 9.0, and was not substantially different from 0 above pH 9.0. These data indicate that P. purpurascens is inefficient in the use of external HCO₃ ⁻ as a carbon source in photosynthesis. Moreover, the photosynthetic rate as a function of external DIC and the highest pH (9.01 ± 0.07) that this species can achieve in a closed system were consistent with a low capacity to use HCO₃ ⁻, in comparison to many other species of seaweeds. The role of external carbonic anhydrase (CA; EC 4.2.1.1) on carbon uptake was investigated by measuring both the HCO₃ ⁻-dependent O₂ evolution and the CO₂ uptake, at pH 5.5 and 8.0, and the rate of pH change in the external medium, in the presence of selected inhibitors of extra- and intracellular CA. Photosynthetic DIC-dependent O₂ evolution was higher at pH 5.5 (where CO₂ is the predominant form of DIC) than at pH 8.0 (where the predominant chemical species is HCO₃ ⁻). Both intra- and extracellular CA activity was detected. Dextran-bound sulfonamide (DBS; a specific inhibitor of extracellular CA) reduced the photosynthetic O₂ evolution and CO₂ uptake at pH 8.0, but there was no effect at pH 5.5. The pH-change rate of the medium, under saturating irradiance, was reduced by DBS. Phyllariopsis purpurascens has a low efficiency in the use of HCO₃ ⁻ as carbon source in photosynthesis; nevertheless, the ion can be used after dehydration, in the external medium, catalyzed by extracellular CA. This mechanism could explain why the photosynthetic rate in situ was higher than that supported solely by the diffusion of CO₂ from seawater. Received: 6 March 1998 / Accepted: 22 June 1998     

State-transitions facilitate robust quantum yields and cause an over-estimation of electron transport in Dunaliella tertiolecta cells held at the CO2 compensation point and re-supplied with DIC

Photosynthetic energy consumption and non-photosynthetic energy quenching processes are inherently linked. Both processes must be controlled by the cell to allow cell maintenance and growth, but also to avoid photodamage. We used the chlorophyte algae Dunaliella tertiolecta to investigate how the interactive regulation of photosynthetic and non-photosynthetic pathways varies along dissolved inorganic carbon (DIC) and photon flux gradients. Specifically, cells were transferred to DIC-deplete media to reach a CO₂ compensation before being re-supplied with DIC at various concentrations and different photon flux levels. Throughout these experiments we monitored and characterized the photophysiological responses using pulse amplitude modulated fluorescence, oxygen evolution, 77 K fluorescence emission spectra, and fast-repetition rate fluorometry. O₂ uptake was not significantly stimulated at DIC depletion, which suggests that O₂ production rates correspond to assimilatory photosynthesis. Fluorescence-based measures of relative electron transport rates (rETRs) over-estimated oxygen-based photosynthetic measures due to a strong state-transitional response that facilitated high effective quantum yields. Adoption of an alternative fluorescence-based rETR calculation that accounts for state-transitions resulted in improved linear oxygen versus rETR correlation. This study shows the extraordinary capacity of D. tertiolecta to maintain stable effective quantum yields by flexible regulation of state-transitions. Uncertainties about the control mechanisms of state-transitions are presented.     

Rates of glycolate synthesis and metabolism during photosynthesis of Euglena and microalgae grown on low CO2

The rate of glycolate excretion in Euglena gracilis Z and some microalgae grown at the atmospheric level of CO₂ was determined using amino-oxyacetate (AOA). The extracellular O₂ concentration was kept at 240 M by bubbling the incubation medium with air. Glycolate, the main excretion product, was excreted by Euglena at 6 mol·h^-1·(mg chlorophyll (Chl))^-1. Excretion depended on the presence of AOA, and was saturated at 1 mM AOA. A substituted oxime formed from glyoxylate and AOA was also excreted. Bicarbonate added at 0.1 mM did not prevent the excretion of glycolate. The excretion of glycolate increased with higher O₂ concentrations in the medium, and was competitively inhibited by much higher concentrations of bicarbonate. Aminooxyacetate also caused excretion of glycolate from the green algae, Chlorella pyrenoidosa, Scenedesmus obliquus and Chlamydomonas reinhardtii grown on air, at the rates of 2–7 mol·h^-1·(mg Chl)^-1 in the presence of 0.2–0.6 mM dissolved inorganic carbon, but the cyanobacterium, Anacystis nidulans, grown in the same way did not excrete glycolate. The efficiency of the CO₂-concentrating mechanism to suppress glycolate formation is discussed on the basis of the magnitude of glycolate formation in these low-CO₂-grown cells.Abbreviations AOA aminooxyacetate - Chl chlorophyll - DIC dissolved inorganic carbon - HPLC high-pressure liquid chromatography - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This is the 16th paper in a series on the metabolism of glycolate in Euglena gracilis. The 15th paper is Yokota et al. (1985c)     

EFFECTS OF CO2 ENRICHMENT ON THE BLOOM‐FORMING CYANOBACTERIUM MICROCYSTIS AERUGINOSA (CYANOPHYCEAE): PHYSIOLOGICAL RESPONSES AND RELATIONSHIPS WITH THE AVAILABILITY OF DISSOLVED INORGANIC CARBON1

Microcystis aeruginosa Kütz. 7820 was cultured at 350 and 700 μL·L ^{? 1} CO₂ to assess the impacts of doubled atmospheric CO₂ concentration on this bloom‐forming cyanobacterium. Doubling of CO₂ concentration in the airflow enhanced its growth by 52%–77%, with pH values decreased and dissolved inorganic carbon (DIC) increased in the medium. Photosynthetic efficiencies and dark respiratory rates expressed per unit chl a tended to increase with the doubling of CO₂. However, saturating irradiances for photosynthesis and light‐saturated photosynthetic rates normalized to cell number tended to decrease with the increase of DIC in the medium. Doubling of CO₂ concentration in the airflow had less effect on DIC‐saturated photosynthetic rates and apparent photosynthetic affinities for DIC. In the exponential phase, CO₂ and HCO₃ ^? levels in the medium were higher than those required to saturate photosynthesis. Cultures with surface aeration were DIC limited in the stationary phase. The rate of CO₂ dissolution into the liquid increased proportionally when CO₂ in air was raised from 350 to 700 μL·L ^{? 1}, thus increasing the availability of DIC in the medium and enhancing the rate of photosynthesis. Doubled CO₂ could enhance CO₂ dissolution, lower pH values, and influence the ionization fractions of various DIC species even when the photosynthesis was not DIC limited. Consequently, HCO₃ ^? concentrations in cultures were significantly higher than in controls, and the photosynthetic energy cost for the operation of CO₂ concentrating mechanism might decrease.     

Circadian photosynthetic reductant flow in the dinoflagellate Lingulodinium is limited by carbon availability

Circadian rhythms are the observed outputs of endogenous daily clocks and are thought to provide a selective advantage to cells adapted to daily light/dark cycles. However, the biochemical links between the clock and the overt rhythms in cell physiology are generally not known. Here, we examine the circadian rhythm in O₂ evolution by cultures of the dinoflagellate Lingulodinium, a rhythm previously ascribed to rhythmic electron flow through photosystem II. We find that O₂ evolution rates increase when CO₂ concentrations are increased, either following addition of DIC or a rapid decrease in culture pH. In medium containing only nitrate as an electron acceptor, O₂ evolution rates mirror the circadian rhythm of nitrate reductase activity in the cells. Furthermore, competition between photosynthetic electron flow to carbon and to nitrate varies in its relative efficiency through the day–night cycle. We also find, using simultaneous and continuous monitoring of pH and O₂ evolution rates over several days, that while culture pH is normally rhythmic, circadian changes in rates of O₂ evolution depend not on the external pH but on levels of internal electron acceptors. We propose that the photosynthetic electron transport rhythm in Lingulodinium is driven by the availability of a reductant sink.     

The Role of External Carbonic Anhydrase in Inorganic Carbon Acquisition by Chlamydomonas reinhardii at Alkaline pH

The role of external carbonic anhydrase in inorganic carbon acquisition and photosynthesis by Chlamydomonas reinhardii at alkaline pH (8.0) was studied. Acetazolamide (50 micromolar) completely inhibited external carbonic anhydrase (CA) activity as determined from isotopic disequilibrium experiments. Under these conditions, photosynthetic rates at low dissolved inorganic carbon (DIC) were far greater than could be maintained by CO₂ supplied from the spontaneous dehydration of HCO₃⁻ thereby showing that C. reinhardii has the ability to utilize exogenous HCO₃⁻. Acetazolamide increased the concentration of DIC required to half-saturate photosynthesis from 38 to 80 micromolar, while it did not affect the maximum photosynthetic rate. External CA activity was also removed from the cell-wall-less mutant (CW-15) by washing. This had no effect on the photosynthetic kinetics of the algae while the addition of acetazolamide to washed cells (CW-15) increased the K_½^DIC from 38 to 80 micromolar. Acetazolamide also caused a buildup of the inorganic carbon pool upon NaHCO₃ addition, indicating that this compound partially inhibited internal CA activity. The effects of acetazolamide on the photosynthetic kinetics of C. reinhardii are likely due to the inhibition of internal rather than a consequence of the inhibition of external CA. Further analysis of the isotopic disequilibrium experiments at saturating concentration of DIC provided evidence consistent with active CO₂ transport by C. reinhardii. The observation that C. reinhardii has the ability to take up both CO₂ and bicarbonate throws into question the role of external CA in the accumulation of DIC in this alga.     

Is the tetrasporophyte of Asparagopsis armata (Bonnemaisoniales) limited by inorganic carbon in integrated aquaculture?1

Seaweeds cultivated in traditional land‐based tank systems usually grow under carbon‐limited conditions and consequently have low production rates, if no costly artificial source of inorganic carbon is supplied. In integrated aquaculture, the fish effluents provide an extra source of dissolved inorganic carbon (DIC) to seaweeds due to fish respiration. To evaluate if the tetrasporophyte of Asparagopsis armata (Harv.) F. Schmitz (the Falkenbergia stage) is carbon limited when cultivated with effluents of a fish (Sparus aurata) farm in southern Portugal, we characterized the DIC forms in the water, assessed the species photosynthetic response to the different DIC concentrations and pH values, and inferred for the presence of a carbonic anhydrase (CA)–mediated mechanism. Results showed that A. armata relies mainly on CO₂ to meet photosynthetic needs. Nevertheless, from pH 7.5 upward, the CO₂ supply to RUBISCO seems to derive also from the external dehydration of HCO₃^– mediated by CA. The contribution of this mechanism was essential for A. armata to attain fully saturated O₂‐evolution rates at the natural seawater DIC concentration (2–2.2 mM) and pH values (～8.0). We revealed in this study that seaweeds cultivated in fish‐farm effluents benefit not only from a rich source of ammonia but also from an important and free source of DIC for their photosynthesis. If supplied at relatively high turnover rates (～100 vol · d^?1), fish‐farm effluents provide enough carbon to maximize the photosynthesis and growth even for species with low affinity for HCO₃^–, avoiding the artificial and costly supply of inorganic carbon to seaweed cultures.     

The Relationship between Ribulose Bisphosphate Concentration, Dissolved Inorganic Carbon (DIC) Transport and DIC-Limited Photosynthesis in the Cyanobacterium Synechococcus leopoliensis Grown at Different Concentrations of Inorganic Carbon

To examine the factors which limit photosynthesis and their role in photosynthetic adaptation to growth at low dissolved inorganic carbon (DIC), Synechococcus leopoliensis was grown at three concentrations (as signified by brackets) of DIC, high (1000-1800 micromolar), intermediate (200-300 micromolar), and low (10-20 micromolar). In all cell types photosynthesis varied from being ribulose bisphosphate (RuBP)-saturated at low external [DIC] to RuBP-limited at high external [DIC]. The maximum rate of photosynthesis (P_max) was achieved when the internal concentration of RuBP fell below the active site density of RuBP carboxylase/oxygenase (Rubisco). At rates of photosynthesis below P_max, photosynthetic capacity was limited by the ability of the cell to transport inorganic carbon and to supply CO₂ to Rubisco. Adaptation to low DIC was reflected by a decrease in the [DIC] required to half-saturate photosynthesis. Simultaneous mass-spectrometric measurement of rates of photosynthesis and DIC transport showed that the initial slope of the photosynthesis versus [DIC] curve is identical to the initial slope of the DIC transport versus [DIC] curve. This provided evidence that the enhanced capacity for DIC transport which occurs upon adaptation to low [DIC] was responsible for the increase in the initial slope of the photosynthesis versus [DIC] curve and therefore the decrease in the half saturation constant of photosynthesis with respect to DIC. Levels of RuBP and in vitro Rubisco activity varied only slightly between high and intermediate [DIC] grown cells but fell significantly (65-70%) in low [DIC] grown cells. Maximum rates of photosynthesis followed a similar pattern with P_max only slightly lower in intermediate [DIC] grown cells than in high [DIC] grown cells, but much lower in low [DIC] grown cells. The changing response of photosynthesis to [DIC] during adaptation to low DIC, may be explained by the interaction between DIC-transport limited and [RuBP]-limited photosynthesis.     

Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts

Photoassimilation of ¹⁴CO₂ by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.

DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO₂ assimilation occurred.

The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator.

     

Photosynthetic characteristics of coccoid marine cyanobacteria

Two strains of marine Synechococcus possessed a much greater potential for photorespiration than other marine algae we have studied. This conclusion was based on the following physiological and biochemical characteristics: a) a light-dependent O₂ inhibition of photosynthetic CO₂ assimilation at atmospheric O₂ concentrations. The degree of inhibition was dependent on the relative concentrations of dissolved O₂ and CO₂, being greatest at 100% O₂ with no extra bicarbonate added to the medium; b) actively photosynthesizing cells had high levels of ribulose-1,5-bisphosphate carboxylase compared with phosphoenolpyruvate carboxylase; ribulose-1,5-bisphosphate oxygenase activities were three times greater than ribulose-1,5-bisphosphate carboxylase activities; c) cells photosynthesizing in 21% O₂, showed significant ¹⁴C-labelling of phosphoglycolate and glycolate and the percentage of total carbon-14 incorporated into these two compounds increased when the O₂ concentration was 100%; d) at 100% O₂, there was a post-illumination enhanced rate of O₂ consumption, which was three times greater than dark respiration, and the rate declined with increasing bicarbonate concentrations. The inhibitory effect of O₂ on photosynthesis did not appear to be solely due to photorespiration, since O₂ inhibition of photosynthetic O₂ evolution was much greater than that of photosynthetic CO₂ assimilation. Also, O₂ inhibition of photosynthetic O₂ evolution declined only slightly with decreasing light intensities, while the inhibition of CO₂ assimilation declined rapidly with decreasing light intensity.