Effect of dissolved inorganic carbon on oxygen evolution and uptake by Chlamydomonas reinhardtii suspensions adapted to ambient and CO2-enriched air

Mass spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ isotopes were used to compare the rates of gross O₂ evolution (E₀), O₂ uptake (U₀) and net O₂ evolution (NET) in relation to different concentrations of dissolved inorganic carbon (DIC) by Chlamydomonas reinhardtii cells grown in air (air-grown), in air enriched with 5% CO₂ (CO₂-grown) and by cells grown in 5% CO₂ and then adapted to air for 6h (air-adapted).At a photon fluence rate (PFR) saturating for photosynthesis (700 mol photons m^-2 s^-1), pH=7.0 and 28°C, U₀ equalled E₀ at the DIC compensation point which was 10M DIC for CO₂-grown and zero for air-grown cells. Both E₀ and U₀ were strongly dependent on DIC and reached DIC saturation at 480 M and 70 M for CO₂-grown and air-grown algae respectively. U₀ increased from DIC compensation to DIC saturation. The U₀ values were about 40 (CO₂-grown), 165 (air-adapted) and 60 mol O₂ mg Chl^-1 h^-1 (air-grown). Above DIC compensation the U₀/E₀ ratios of air-adapted and air-grown algae were always higher than those of CO₂-grown cells. These differences in O₂ exchange between CO₂- and air-grown algae seem to be inducable since air-adapted algae respond similarly to air-grown cells.For all algae, the rates of dark respiratory O₂ uptake measured 5 min after darkening were considerably lower than the rates of O₂ uptake just before darkening. The contribution of dark respiration, photorespiration and the Mehler reaction to U₀ is discussed and the energy requirement of the inducable CO₂/HCO₃ ^- concentrating mechanism present in air-adapted and air-grown C. reinhardtii cells is considered.Abbreviations DIC dissolved inorganic carbon - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - E₀ rate of photosynthetic gross O₂ evolution - PCO photosynthetic carbon oxidation - PFR photon fluence rate - PS I photosystem I - PS II photosystem II - U₀ rate of O₂ uptake in the light - MS mass spectrometer     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

CO(2) and O(2) Exchange in Two Mosses, Hypnum cupressiforme and Dicranum scoparium

Photosynthetic CO₂ and O₂ exchange was studied in two moss species, Hypnum cupressiforme Hedw. and Dicranum scoparium Hedw. Most experiments were made during steady state of photosynthesis, using ¹⁸O₂ to trace O₂ uptake. In standard experimental conditions (photoperiod 12 h, 135 micromoles photons per square meter per second, 18°C, 330 microliters per liter CO₂, 21% O₂) the net photosynthetic rate was around 40 micromoles CO₂ per gram dry weight per hour in H. cupressiforme and 50 micromoles CO₂ per gram dry weight per hour in D. scoparium. The CO₂ compensation point lay between 45 and 55 microliters per liter CO₂ and the enhancement of net photosynthesis by 3% O₂versus 21% O₂ was 40 to 45%. The ratio of O₂ uptake to net photosynthesis was 0.8 to 0.9 irrespective of the light intensity. The response of net photosynthesis to CO₂ showed a high apparent K_m (CO₂) even in nonsaturating light. On the other hand, O₂ uptake in standard conditions was not far from saturation. It could be enhanced by only 25% by increasing the O₂ concentration (saturating level as low as 30% O₂), and by 65% by decreasing the CO₂ concentration to the compensation point. Although O₂ is a competitive inhibitor of CO₂ uptake it could not replace CO₂ completely as an electron acceptor, and electron flow, expressed as gross O₂ production, was inhibited by both high O₂ and low CO₂ levels. At high CO₂, O₂ uptake was 70% lower than the maximum at the CO₂ compensation point. The remaining activity (30%) can be attributed to dark respiration and the Mehler reaction.     

Zeaxanthin and the Induction and Relaxation Kinetics of the Dissipation of Excess Excitation Energy in Leaves in 2% O(2), 0% CO(2)

The relationship between the carotenoid zeaxanthin, formed by violaxanthin de-epoxidation, and nonphotochemical fluorescence quenching (q_NP) in the light was investigated in leaves of Glycine max during a transient from dark to light in 2% O₂, 0% CO₂ at 100 to 200 micromoles of photons per square meter per second. (a) Up to a q_NP (which can vary between 0 and 1) of about 0.7, the zeaxanthin content of leaves was linearly correlated with q_NP as well as with the rate constant for radiationless energy dissipation in the antenna chlorophyll (k_D). Beyond this point, at very high degrees of fluorescence quenching, only k_D was directly proportional to the zeaxanthin content. (b) The relationship between zeaxanthin and k_D was quantitatively similar for the rapidly relaxing quenching induced in 2% O₂, 0% CO₂ at 200 micromoles of photons per square meter per second and for the sustained quenching induced by long-term exposure of Nerium oleander to drought in high light (B Demmig, K Winter, A Krüger, F-C Czygan [1988] Plant Physiol 87: 17-24). These findings suggest that the same dissipation process may be induced by very different treatments and that this particular dissipation process can have widely different relaxation kinetics. (c) A rapid induction of strong nonphotochemical fluorescence quenching within about 1 minute was observed exclusively in leaves which already contained a background level of zeaxanthin.     

Glycolate Metabolism and Excretion by Chlamydomonas reinhardtii

The flux of glycolate through the C₂ pathway in Chlamydomonas reinhardtii was estimated after inhibition of the pathway with aminooxyacetate (AOA) or aminoacetonitrile (AAN) by measurement of the accumulation of glycolate and glycine. Cells grown photoautotrophically in air excreted little glycolate except in the presence of 2 mm AOA when they excreted 5 micromoles glycolate per hour per milligram clorophyll. Cells grown on high CO₂ (1-5%) when transferred to air produced three times as much glycolate, with half of the glycolate metabolized and half excreted. The lower amount of glycolate produced by the air-grown cells reflects the presence of a CO₂ concentrating mechanism which raises the internal CO₂ level and decreases the ribulose-1,5-bisP oxygenase reaction for glycolate production. Despite the presence of the CO₂ concentrating mechanism, there was still a significant amount of glycolate produced and metabolized by air-grown Chlamydomonas. The capacity of these cells to metabolize between 5 and 10 micromoles of glycolate per hour per milligram chlorophyll was confirmed by measuring the biphasic uptake of added labeled glycolate. The initial rapid (<10 seconds) phase represented uptake of glycolate; the slow phase represented the metabolism of glycolate. The rates of glycolate metabolism were in agreement with those determined using the C₂-cycle inhibitors during CO₂ fixation.     

Relationships between Photosynthetically Active Radiation, Nocturnal Acid Accumulation, and CO(2) Uptake for a Crassulacean Acid Metabolism Plant, Opuntia ficus-indica

The influences of photosynthetically active radiation (PAR) and water status on nocturnal Crassulacean acid metabolism (CAM) were quantitatively examined for a widely cultivated cactus, Opuntia ficus-indica (L.) Miller. When the total daily PAR was maintained at 10 moles photons per square meter per day but the instantaneous PAR level varied, the rate of nocturnal H⁺ accumulation (tissue acidification) became 90% saturated near 700 micromoles per square meter per second, a PAR level typical for similar light saturation of C₃ photosynthesis. The total nocturnal H⁺ accumulation and CO₂ uptake reached 90% of maximum for a total daily PAR of about 22 moles per square meter per day. Light compensation occurred near 0 moles per square meter per day for nocturnal H⁺ accumulation and 4 moles per square meter per day for CO₂ uptake. Above a total daily PAR of 36 moles per square meter per day or for an instantaneous PAR of 1150 micromoles per square meter per second for more than 6 hours, the nocturnal H⁺ accumulation actually decreased. This inhibition, which occurred at PAR levels just above those occurring in the field, was accompanied by a substantial decrease in chlorophyll content over a 1-week period.

A minimum ratio of H⁺ accumulated to CO₂ taken up of 2.5 averaged over the night occurred for a total daily PAR of 31 moles per square meter per day under wet conditions. About 2 to 6 hours into the night under such conditions, a minimum H⁺-to-CO₂ ratio of 2.0 was observed. Under progressively drier conditions, both nocturnal H⁺ accumulation and CO₂ uptake decreased, but the H⁺-to-CO₂ ratio increased. A ratio of two H⁺ per CO₂ is consistent with the H⁺ production accompanying the conversion of starch to malic acid, and it apparently occurs for O. ficus-indica when CAM CO₂ uptake is strongly favored over respiratory activity.

     

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Response to Light Intensity and CO(2) in the C(3) Annuals Chenopodium album L. and Phaseolus vulgaris L

The light and CO₂ response of (a) photosynthesis, (b) the activation state and total catalytic efficiency (k_cat) of ribulose-1,5-bisphosphate carboxylase (rubisco), and (c) the pool sizes of ribulose 1,5-bisphosphate, (RuBP), ATP, and ADP were studied in the C₃ annuals Chenopodium album and Phaseolus vulgaris at 25°C. The initial slope of the photosynthetic CO₂ response curve was dependent on light intensity at reduced light levels only (less than 450 micromoles per square meter per second in C. album and below 200 micromoles per square meter per second in P. vulgaris). Modeled simulations indicated that the initial slope of the CO₂ response of photosynthesis exhibited light dependency when the rate of RuBP regeneration limited photosynthesis, but not when rubisco capacity limited photosynthesis. Measured observations closely matched modeled simulations. The activation state of rubisco was measured at three light intensities in C. album (1750, 550, and 150 micromoles per square meter per second) and at intercellular CO₂ partial pressures (C₁) between the CO₂ compensation point and 500 microbars. Above a C₁ of 120 microbars, the activation state of rubisco was light dependent. At light intensities of 550 and 1750 micromoles per square meter per second, it was also dependent on C₁, decreasing as the C₁ was elevated above 120 microbars at 550 micromoles per square meter per second and above 300 microbars at 1750 micromoles per square meter per second. The pool size of RuBP was independent of C₁ only under conditions when the activation state of rubisco was dependent on C₁. Otherwise, RuBP pool sizes increased as C₁ was reduced. ATP pools in C. album tended to increase as C₁ was reduced. In P. vulgaris, decreasing C₁ at a subsaturating light intensity of 190 micromoles per square meter per second increased the activation state of rubisco but had little effect on the k_cat. These results support modelled simulations of the rubisco response to light and CO₂, where rubisco is assumed to be down-regulated when photosynthesis is limited by the rate of RuBP regeneration.     

Participation of Photosynthesis in Floral Induction of the Long Day Plant Anagallis arvensis L

The saturating photon flux density (400 to 700 nanometers) for induction of flowering of the long day plant Anagallis arvensis L. was 1,900 micromoles per square meter per second (6,000 foot-candles) when an 8-hour daylength was extended to 24 hours by a single period of supplementary irradiation. The saturating photon flux density for photosynthetic CO₂ uptake during the same single supplementary light period was lower, at about 1,000 to 650 micromoles per square meter per second (3,000 to 2,000 foot-candles).

The per cent flowering and mean number of floral buds per plant were significantly reduced when the light extension treatment was given in CO₂-free air, and glucose (10 kilograms per cubic meter in water) relieved this effect. Glucose solution also significantly increased flowering of plants given supplementary light treatment in atmospheric air under a photon flux density of 80 micromoles per square meter per second. Increasing the CO₂ concentration to 1.27 grams per cubic meter of CO₂ in air during the supplementary light period did not increase flowering.

It is concluded that high photon flux densities promote flowering of Anagallis through both increased photosynthesis and the photomorphogenic action of high irradiance.

     

Photosynthetic and Respiratory Activity of Fruiting Forms within the Cotton Canopy

The supply of photosynthates by leaves for reproductive development in cotton (Gossypium hirsutum L.) has been extensively studied. However, the contribution of assimilates derived from the fruiting forms themselves is inconclusive. Field experiments were conducted to document the photosynthetic and respiratory activity of cotton leaves, bracts, and capsule walls from anthesis to fruit maturity. Bracts achieved peak photosynthetic rates of 2.1 micromoles per square meter per second compared with 16.5 micromoles per square meter per second for the subtending leaf. However, unlike the subtending leaf, the bracts did not show a dramatic decline in photosynthesis with increased age, nor was their photosynthesis as sensitive as leaves to low light and water-deficit stress. The capsule wall was only a minor site of ¹⁴CO₂ fixation from the ambient atmosphere. Dark respiration by the developing fruit averaged −18.7 micromoles per square meter per second for 6 days after anthesis and declined to −2.7 micromoles per square meter per second after 40 days. Respiratory loss of CO₂ was maximal at −158 micromoles CO₂ per fruit per hour at 20 days anthesis. Diurnal patterns of dark respiration for the fruit were age dependent and closely correlated with stomatal conductance of the capsule wall. Stomata on the capsule wall of young fruit were functional, but lost this capacity with increasing age. Labeled ¹⁴CO₂ injected into the fruit interior was rapidly assimilated by the capsule wall in the light but not in the dark, while fiber and seed together fixed significant amounts of ¹⁴CO₂ in both the light and dark. These data suggest that cotton fruiting forms, although sites of significant respiratory CO₂ loss, do serve a vital role in the recycling of internal CO₂ and therein, function as important sources of assimilate for reproductive development.     

Developmental Changes in Photosynthetic Gas Exchange in the Polyol-Synthesizing Species, Apium graveolens L. (Celery)

Developmental changes in photosynthetic gas exchange were investigated in the mannitol synthesizing plant celery (Apium graveolens L. `Giant Pascal'). Greenhouse-grown plants had unusually high photosynthetic rates for a C₃ plant, but consistent with field productivity data reported elsewhere for this plant. In most respects, celery exhibited typical C₃ photosynthetic characteristics; light saturation occurred at 600 micromoles photons per square meter per second, with a broad temperature optimum, peaking at 26°C. At 2% O₂, photosynthesis was enhanced 15 to 25% compared to rates at 21% O₂. However, celery had low CO₂ compensation points, averaging 7 to 20 microliters per liter throughout the canopy. Conventional mechanisms for concentrating CO₂ were not detectable.     

Light-Dependent Oxygen Uptake, Glycolate, and Ammonia Release in l-Methionine Sulfoximine-Treated Chlamydomonas

Glycolate and ammonia excretion plus oxygen exchanges were measured in the light in l-methionine-dl-sulfoximine treated air-grown Chlamydomonas reinhardii. At saturating CO₂ (between 600 and 700 microliters per liter CO₂) neither glycolate nor ammonia were excreted, whereas at the CO₂ compensation concentration (<10 microliters per liter CO₂) treated algae excreted both glycolate and ammonia at rates of 37 and 59 nanomoles per minute per milligram chlorophyll, respectively. From the excretion values we calculate the amount of O₂ consumed through the glycolate pathway. The calculated value was not significantly different from the component of O₂ uptake sensitive to CO₂ obtained from the difference between O₂ uptake of the CO₂ compensation point and at saturating CO₂. This component was about 40% of stationary O₂ uptake measured at the CO₂ compensation point. From these data we conclude that glyoxylate decarboxylation in air-grown Chlamydomonas represents a minor pathway of metabolism even in conditions where amino donors are deficient and that processes other than glycolate pathway are responsible for the O₂ uptake insensitive to CO₂.     

Effect of the Long-Term Elevation of CO(2) Concentration in the Field on the Quantum Yield of Photosynthesis of the C(3) Sedge, Scirpus olneyi

CO₂ concentration was elevated throughout 3 years around stands of the C₃ sedge Scirpus olneyi on a tidal marsh of the Chesapeake Bay. The hypothesis that tissues developed in an elevated CO₂ atmosphere will show an acclimatory decrease in photosynthetic capacity under light-limiting conditions was examined. The absorbed light quantum yield of CO₂ uptake (ø_abs and the efficiency of photosystem II photochemistry were determined for plants which had developed in open top chambers with CO₂ concentrations in air of 680 micromoles per mole, and of 351 micromoles per mole as controls. An Ulbricht sphere cuvette incorporated into an open gas exchange system was used to determine ø_abs and a portable chlorophyll fluorimeter was used to estimate the photochemical efficiency of photosystem II. When measured in an atmosphere with 10 millimoles per mole O₂ to suppress photorespiration, shoots showed a ø_abs of 0.093 ± 0.003, with no statistically significant difference between shoots grown in elevated or control CO₂ concentrations. Efficiency of photosystem II photochemistry was also unchanged by development in an elevated CO₂ atmosphere. Shoots grown and measured in 680 micromoles per mole of CO₂ in air showed a ø_abs of 0.078 ± 0.004 compared with 0.065 ± 0.003 for leaves grown and measured in 351 micromoles per mole CO₂ in air; a highly significant increase. In accordance with the change in ø_abs, the light compensation point of photosynthesis decreased from 51 ± 3 to 31 ± 3 micro-moles per square meter per second for stems grown and measured in 351 and 680 micromoles per mole of CO₂ in air, respectively. The results suggest that even after 3 years of growth in elevated CO₂, there is no evidence of acclimation in capacity for photosynthesis under light-limited conditions which would counteract the stimulation of photosynthetic CO₂ uptake otherwise expected through decreased photorespiration.     

Whole Leaf Carbon Exchange Characteristics of Phosphate Deficient Soybeans (Glycine max L.)

Low phosphate nutrition results in increased chlorophyll fluorescence, reduced photosynthetic rate, accumulation of starch and sucrose in leaves, and low crop yields. This study investigated physiological responses of soybean (Glycine max [L.] Merr.) leaves to low inorganic phosphate (Pi) conditions. Responses of photosynthesis to light and CO₂ were examined for leaves of soybean grown at high (0.50 millimolar) or low (0.05 millimolar) Pi. Leaves of low Pi plants exhibited paraheliotropic orientation on bright sunny days rather than the normal diaheliotropic orientation exhibited by leaves of high Pi soybeans. Leaves of plants grown at high Pi had significantly higher light saturation points (1000 versus 630 micromole photons [400-700 nanometers] per square meter per second) and higher apparent quantum efficiency (0.062 versus 0.044 mole CO₂ per mole photons) at ambient (34 pascals) CO₂ than did low Pi leaves, yet stomatal conductances were similar. High Pi leaves also had significantly higher carboxylation efficiency (2.90 versus 0.49 micromole CO₂ per square meter per second per pascal), a lower CO₂ compensation point (6.9 versus 11.9 pascals), and a higher photosynthetic rate at 34 pascals CO₂ (19.5 versus 6.7 micromoles CO₂ per square meter per second) than did low Pi leaves. Soluble protein (0.94 versus 0.73 milligram per square centimeter), ribulose-1,5-bisphosphate carboxylase/oxygenase content (0.33 versus 0.25 milligram per square centimeter), and ribulose-1,5-bisphosphate carboxylase/oxygenase specific activity (25.0 versus 16.7 micromoles per square meter per second) were significantly greater in leaves of plants in the high Pi treatment. The data indicate that Pi stress alters the plant's CO₂ reduction characteristics, which may in turn affect the plant's capacity to accommodate normal radiation loads.     

Photorespiration in Air and High CO(2)-Grown Chlorella pyrenoidosa

Oxygen inhibition of photosynthesis and CO₂ evolution during photorespiration were compared in high CO₂-grown and air-grown Chlorella pyrenoidosa, using the artificial leaf technique at pH 5.0. High CO₂ cells, in contrast to air-grown cells, exhibited a marked inhibition of photosynthesis by O₂, which appeared to be competitive and similar in magnitude to that in higher C₃ plants. With increasing time after transfer to air, the photosynthetic rate in high CO₂ cells increased while the O₂ effect declined. Photorespiration, measured as the difference between ¹⁴CO₂ and ¹²CO₂ uptake, was much greater and sensitive to O₂ in high CO₂ cells. Some CO₂ evolution was also present in air-grown algae; however, it did not appear to be sensitive to O₂. True photosynthesis was not affected by O₂ in either case. The data indicate that the difference between high CO₂ and air-grown algae could be attributed to the magnitude of CO₂ evolution. This conclusion is discussed with reference to the oxygenase reaction and the control of photorespiration in algae.     

Crassulacean Acid Metabolism and Photochemical Efficiency of Photosystem II in the Adaxial and Abaxial Parts of the Succulent Leaves of Kalanchoë daigremontiana Grown at Four Photon Flux Densities

Kalanchoë daigremontiana, a species possessing crassulacean acid metabolism, was grown at four photon flux densities (1300, 400, 60, and 25 micromole photons per square meter per second). In leaves which had developed at 1300 and 400 micromole photons per square meter per second, CO₂ was mainly incorporated through the lower, shaded leaf surfaces, and the chlorenchyma adjacent to the lower surfaces showed a higher degree of nocturnal acid synthesis than the chlorenchyma adjacent to the upper surfaces. In leaves acclimated to 60 and 25 micromole photons per square meter per second, the gradient in CAM activity was reversed, i.e. more CO₂ was taken up through the upper than through the lower surfaces and nocturnal acidification was higher in the tissue next to the upper surfaces. Total net carbon gain and total nocturnal acid synthesis were highest in leaves which had developed at 400 micromole photons per square meter per second. Chlorophyll content was markedly reduced in leaves which had developed at 1300 micromole photons per square meter per second, especially in the exposed adaxial parts. There was also a sustained reduction in photosystem II photochemical efficiency as indicated by measurements of the ratio of variable over maximum chlorophyll a fluorescence. These findings suggest that, at high growth photon flux densities, the reduced activity of the exposed portions of these succulent leaves is caused by (a) the adverse effects of excess light, (b) together with a genotypic component which favors CO₂ uptake and acid synthesis in the abaxial (lower) leaf parts even when light is not or only marginally excessive. This latter component is predominant at medium photon flux densities, e.g. at 400 micromole photons per square meter per second. It becomes overridden, however, under conditions of deep shade when strongly reduced light levels in the abaxial parts of the leaf chlorenchyma severely limit photosynthesis.     

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

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO2

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO₂ concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1–4 days. In contrast, C. littorale cells which had been grown in 5% CO₂ (5% CO₂-grown cells) could grow in 40% CO₂ without any lag period. When air-grown cells were transferred to 40% CO₂, the quantum efficiency of PS II (Φ_II) decreased greatly, while no decrease in Φ_II was apparent when the 5% CO₂-grown cells were transferred to 40% CO₂. In contrast to air-grown cells, 5% CO₂-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO₂-grown cells to air, photosynthetic susceptibility to 40% CO₂ was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO₂. The intracellular pH value (pH_i) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO₂ for 1–2 h, but no such decrease in pH_i was apparent in the presence of EZA. Both air- and 5% CO₂-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO₂ concentrations, grew in 40% CO₂ without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO₂. No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glycolate Metabolism in Low and High CO(2)-Grown Chlorella pyrenoidosa and Pavlova lutheri as Determined by O-Labeling

Photorespiration in Chlorella pyrenoidosa Chick. was assayed by measuring ¹⁸O-labeled intermediates of the glycolate pathway. Glycolate, glycine, serine, and excreted glycolate were isolated and analyzed on a gas chromatograph/mass spectrometer to determine isotopic enrichment. Rates of glycolate synthesis were determined from ¹⁸O-labeling kinetics of the intermediates, pool sizes, derived rate equations, and nonlinear regression techniques. Glycolate synthesis was higher in high CO₂-grown cells than in air-grown cells when both were assayed under the same O₂ and CO₂ concentrations. Synthesis of glycolate, for both types of cells, was stimulated by high O₂ levels and inhibited by high CO₂ levels. Glycolate synthesis in 1.5% CO₂-grown Chlorella, when exposed to a 0.035% CO₂ atmosphere, increased from about 41 to 86 nanomoles per milligram chlorophyll per minute when the O₂ concentration was increased from 21% to 40%. Glycolate synthesis in air-grown cells increased from 2 to 6 nanomoles per milligram chlorophyll per minute under the same gas levels. Synthesis was undetectable when either the O₂ concentration was lowered to 2% or the CO₂ concentration was raised to 1.5%. Glycolate excretion was also sensitive to O₂ and CO₂ concentrations in 1.5% CO₂-grown cells and the glycolate that was excreted was ¹⁸O-labeled. Air-grown cells did not excrete glycolate under any experimental condition. Indirect evidence indicated that glycolate may be excreted as a lactone in Chlorella. Photorespiratory ¹⁸O-labeling kinetics were determined for Pavlova lutheri, which unlike Chlorella and higher plants did not directly synthesize glycine and serine from glycolate. This alga did excrete a significant proportion of newly synthesized glycolate into the media.     

Photosynthetic and stomatal responses of spinach leaves to salt stress

The gas exchange of spinach plants, salt-stressed by adding NaCl to the nutrient solution in increments of 25 millimolar per day to a final concentration of 200 millimolar, was studied 3 weeks after starting NaCl treatment. Photosynthesis became light saturated at 1100 to 1400 micromoles per square meter per second in salt-treated plants and at approximately 2000 micromoles per square meter per second in control plants. Photosynthetic capacity of the mesophyll measured as a function of intercellular partial pressure of CO₂ at the light intensity prevailing during growth and at light saturation were both decreased in the salttreated plants. The CO₂ compensation points and relative enhancements of photosynthesis at low O₂ were not affected by salinity. The lower photosynthetic rates in salt-treated leaves at 450 micromoles per square meter per second were associated with a 70% reduction in stomatal conductance and low intercellular CO₂ (219 microbars; cf. 285 microbars for controls). Increasing photon flux density to light saturation extended the linear portions of the CO₂ response curves, increased stomatal conductances, increased intercellular CO₂ in the salt-treated plants, but lowered it in controls, and accentuated differences in photosynthetic rate (area basis) between the treatments.

Leaves from salt-treated plants were thicker but contained about 73% of the chlorophyll per unit area of control plants. When photosynthetic rates were expressed on a chlorophyll basis there was no difference in initial slope of assimilation versus intercellular CO₂ between treatments. Photosynthetic rates (chlorophyll basis) at light saturation differed only by 20% which was also observed earlier with isolated, intact chloroplasts (Robinson et al. 1983 Plant Physiol 73: 238-242).

Measurement of carbon isotope ratio revealed less discrimination against ¹³C with salt treatment and confirmed the persistence of low intercellular partial pressures of CO₂ during plant growth. The development of a thicker leaf with less chlorophyll per unit area during salt treatment permitted stomatal conductance and intercellular partial pressure of CO₂ to decline without restricting photosynthesis and had the benefit of greatly increasing water use efficiency.

     

Inorganic-carbon uptake by a small-celled strain of Stichococcus bacillaris

Air-grown cells of a marine, small-celled (2 m diameter) strain of Stichococcus bacillaris contained appreciable carbonic-anhydrase activity but this was repressed when cells were grown on air enriched with 5% (v/v) CO₂. Assay of carbonic-anhydrase activity using intact cells and cell extracts showed all activity was intracellular in this Stichococcus strain. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution at pH 5.0, where CO₂ is the predominant form of inorganic carbon, showed that the concentration of inorganic carbon required for half-maximal rate of photosynthetic O₂ evolution [K_0.5(CO₂)] was 4.0 M for both air- and CO₂-grown cells. At pH 8.3 the K_0.5(CO₂) was 0.3 mM for air-grown and 0.6 mM for CO₂-grown cells. Sodium ions did not enhance bicarbonate utilization. Measurement of the internal inorganic-carbon pool (HCO ₃ ^– +CO₂) by the silicone-oil-layer centrifugal filtering technique showed that air- and CO₂-grown cells were able to concentrate inorganic carbon up to 20-fold in relation to the external medium at pH 5.0 but not at pH 8.3. In this alga the high affinity for CO₂ and inorganic-carbon accumulation in CO₂- and air-grown cells results from active CO₂ transport that is not dependent on carbonic-anhydrase activity.Abbreviation Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid