Acclimation to High CO(2) in Bean : Carbonic Anhydrase and Ribulose Bisphosphate Carboxylase

Young bean plants (Phaseolus vulgaris L. cv Seafarer) grew faster in air enriched with CO₂ (1200 microliters per liter) than in ambient CO₂ (330 microliters per liter). However, by 7 days when increases in overall growth (dry weight, leaf area) were visible, there was a significant decline (about 25%) in the leaf mineral content (N, P, K, Ca, Mg) and a drop in the activity of two enzymes of carbon fixation, carbonic anhydrase and ribulose 1,5-bisphosphate (RuBP) carboxylase under high CO₂. Although the activity of neither enzyme was altered in young, expanding leaves during the acclimation period, in mature leaves the activity of carbonic anhydrase was reduced 95% compared with a decline of 50% in ambient CO₂. The drop in RuBP carboxylase was less extreme with 40% of the initial activity retained in the high CO₂ compared with 50% in the ambient atmosphere. While CO₂ enrichment might alter the flow of carbon into the glycolate pathway by modifying the activities of carbonic anhydrase or RuBP carboxylase, there is no early change in the ability of photosynthetic tissue to oxidize glycolate to CO₂.     

Acclimation to High CO(2) in Monoecious Cucumbers : II. Carbon Exchange Rates, Enzyme Activities, and Starch and Nutrient Concentrations

Carbon exchange capacity of cucumber (Cucumis sativus L.) germinated and grown in controlled environment chambers at 1000 microliters per liter CO₂ decreased from the vegetative growth stage to the fruiting stage, during which time capacity of plants grown at 350 microliters per liter increased. Carbon exchange rates (CERs) measured under growth conditions during the fruiting period were, in fact, lower in plants grown at 1000 microliters per liter CO₂ than those grown at 350. Progressive decreases in CERs in 1000 microliters per liter plants were associated with decreasing stomatal conductances and activities of ribulose bisphosphate carboxylase and carbonic anhydrase. Leaf starch concentrations were higher in 1000 microliters per liter CO₂ grown-plants than in 350 microliters per liter grown plants but calcium and nitrogen concentrations were lower, the greatest difference occurring at flowering. Sucrose synthase and sucrose-P-synthase activities were similar in 1000 microliters per liter compared to 350 microliters per liter plants during vegetative growth and flowering but higher in 350 microliters per liter plants at fruiting. The decreased carbon exchange rates observed in this cultivar at 1000 microliters per liter CO₂ could explain the lack of any yield increase (MM Peet 1986 Plant Physiol 80: 59-62) when compared with plants grown at 350 microliters per liter.     

Carbonyl sulfide: an inhibitor of inorganic carbon transport in cyanobacteria

Cells of a high CO₂-requiring mutant (E₁) and wild type of Synechococcus PCC7942 were incubated with COS in the light, then suspended in COS-free medium and their CO₂ exchange was measured using an open gas-analysis system under the conditions where photosynthetic CO₂ fixation is inhibited. When the suspension of cells untreated with COS was illuminated, the rate of CO₂ uptake was high and addition of carbonic anhydrase during illumination released a large amount of CO₂ from the medium into the gas phase. The COS treatment in the light markedly reduced the rate of CO₂ uptake by the cells and the amount of CO₂ released by carbonic anhydrase. Incubation of cells with COS in the dark had no effect on the CO₂-exchange profile. The COS concentration required for 50% inhibition of CO₂ uptake was about 25 micromolar when the concentration of inorganic carbon (C_i) in the medium was 60 micromolar; higher C_i concentrations reduced the inhibitory effect of COS. Measurement of C_i uptake in E₁ cells by a silicone oil centrifugation method also indicated marked reduction of the activities of ¹⁴CO₂ and H¹⁴CO₃⁻ uptake in the cells treated with COS in the light. The results demonstrated that COS is a potent inhibitor of C_i transport.     

Zinc deficiency, carbonic anhydrase, and photosynthesis in leaves of spinach

A shortage in the zinc supply to spinach (Spinacia oleracea L.) drastically reduced carbonic anhydrase levels with little effect on net CO₂ uptake per unit leaf area, except with the most severe zinc stresses. Under these conditions, carbonic anhydrase was below 10% and photosynthesis 60 to 70% of the control levels. When photosynthesis was measured at a range of CO₂ supply levels, zinc-deficient leaves were less efficient at 300 to 350 microliters per liter CO₂ and above, but the same as controls at lower CO₂ levels. This suggests that carbonic anhydrase does not affect the diffusion of CO₂, and that the effect of zinc deficiency was on the photosynthetic process itself. Our evidence does not support the hypothesis that carbonic anhydrase has some role in facilitating the supply of CO₂ to the sites of carboxylation within the chloroplast.     

Photosynthesis and Activation of Ribulose Bisphosphate Carboxylase in Wheat Seedlings : Regulation by CO(2) and O(2)

Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO₂ to activate the enzyme, changes in CO₂ between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO₂ levels and 21% O₂ or 1% or less O₂, the levels of ribulose bisphosphate were high and not limiting for CO₂ fixation. With high leaf ribulose bisphosphate, the K_act(CO₂) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO₂ and O₂, activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex.

The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg²⁺ and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg²⁺ and pH most likely determine the ratio of inactive enzyme-RuBP to active enzyme-CO₂-Mg²⁺-RuBP forms. Higher irradiances favor more optimal Mg²⁺ and pH, with greater activation of the carboxylase and increased photosynthesis.

     

Acclimation of Two Tomato Species to High Atmospheric CO(2): II. Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase

Lycopersicon esculentum Mill. cv Vedettos and Lycopersicon chmielewskii Rick, LA 1028, were exposed to two CO₂ concentrations (330 or 900 microliters per liter) for 10 weeks. The elevated CO₂ concentrations increased the initial ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity of both species for the first 5 weeks of treatment but the difference did not persist during the last 5 weeks. The activity of Mg²⁺-CO₂-activated Rubisco was higher in 900 microliters per liter for the first 2 weeks but declined sharply thereafter. After 10 weeks, leaves grown at 330 microliters per liter CO₂ had about twice the Rubisco activity compared with those grown at 900 microliters per liter CO₂. The two species showed the same trend to Rubisco declines under high CO₂ concentrations. The percent activation of Rubisco was always higher under high CO₂. The phosphoenolpyruvate carboxylase (PEPCase) activity measured in tomato leaves averaged 7.9% of the total Rubisco. PEPCase showed a similar trend with time as the initial Rubisco but with no significant difference between nonenriched and CO₂-enriched plants. Long-term exposure of tomato plants to high CO₂ was previously shown to induce a decline of photosynthetic efficiency. Based on the current study and on previous results, we propose that the decline of activated Rubisco is the main cause of the acclimation of tomato plants to high CO₂ concentrations.     

A Model for HCO(3) Accumulation and Photosynthesis in the Cyanobacterium Synechococcus sp: Theoretical Predictions and Experimental Observations

A simple model based on HCO₃⁻ transport has been developed to relate photosynthesis and inorganic carbon fluxes for the marine cyanobacterium, Synechococcus sp. Nägeli (strain RRIMP N1). Predicted relationships between inorganic carbon transport, CO₂ fixation, internal carbonic anhydrase activity, and leakage of CO₂ out of the cell, allow comparisons to be made with experimentally obtained data. Measurements of inorganic carbon fluxes and internal inorganic carbon pool sizes in these cells were made by monitoring time-courses of CO₂ changes (using a mass spectrometer) during light/dark transients. At just saturating CO₂ conditions, total inorganic carbon transport did not exceed net CO₂ fixation by more than 30%. This indicates CO₂ leakage similar to that estimated for C₄ plants.

For this leakage rate, the model predicts the cell would need a conductance to CO₂ of around 10⁻⁵ centimeters per second. This is similar to estimates made for the same cells using inorganic carbon pool sizes and CO₂ efflux measurements. The model predicts that carbonic anhydrase is necessary internally to allow a sufficiently fast rate of CO₂ production to prevent a large accumulation of HCO₃⁻. Intact cells show light stimulated carbonic anhydrase activity when assayed using ¹⁸O-labeled CO₂ techniques. This is also supported by low but detectable levels of carbonic anhydrase activity in cell extracts, sufficient to meet the requirements of the model.

     

The Regulation of Photosynthesis in Leaves of Field-Grown Spring Wheat (Triticum aestivum L., cv Albis) at Different Levels of Ozone in Ambient Air

Wheat (Triticum aestivum L. cv Albis) was grown in open-top chambers in the field and fumigated daily with charcoal-filtered air (0.015 microliters per liter O₃), nonfiltered air (0.03 microliters per liter O₃), and air enriched with either 0.07 or 0.10 microliters per liter ozone (seasonal 8 hour/day [9 am-5 pm] mean ozone concentration from June 1 until July 10, 1987). Photosynthetic ¹⁴CO₂ uptake was measured in situ. Net photosynthesis, dark respiration, and CO₂ compensation concentration at 2 and 21% O₂ were measured in the laboratory. Leaf segments were freeze-clamped in situ for the determination of the steady state levels of ribulose 1,5-bisphosphate, 3-phosphoglycerate, triose-phosphate, ATP, ADP, AMP, and activity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. Photosynthesis of flag leaves was highest in filtered air and decreased in response to increasing mean ozone concentration. CO₂ compensation concentration and the ratio of dark respiration to net photosynthesis increased with ozone concentration. The decrease in photosynthesis was associated with a decrease in chlorophyll, soluble protein, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate, and adenylates. No decrease was found for triose-phosphate and 3-phosphoglycerate. The ratio of ATP to ADP and of triosephosphate to 3-phosphoglycerate were increased suggesting that photosynthesis was limited by pentose phosphate reductive cycle activity. No limitation occurred due to decreased access of CO₂ to photosynthetic cells since the decrease in stomatal conductance with increasing ozone concentration did not account for the decrease in photosynthesis. Ozonestressed leaves showed an increased degree of activation of ribulose bisphosphate carboxylase/oxygenase and a decreased ratio of ribulose bisphosphate to initial activity of ribulose bisphosphate carboxylase/oxygenase. Nevertheless, it is suggested that photosynthesis in ozone stressed leaves is limited by ribulose bisphosphate carboxylation possibly due to an effect of ozone on the catalysis by ribulose bisphosphate carboxylase/oxygenase.     

Reversibility of Photosynthetic Inhibition in Cotton after Long-Term Exposure to Elevated CO(2) Concentrations

Cotton (Gossypium hirsutum L. cv Stoneville 213) was grown at 350 and 1000 microliters per liter CO₂. The plants grown at elevated CO₂ concentrations contained large starch pools and showed initial symptoms of visible physical damage. Photosynthetic rates were lower than expected based on instantaneous exposure to high CO₂.

A group of plants grown at 1000 microliters per liter CO₂ was switched to 350 microliters per liter CO₂. Starch pools and photosynthetic rates were monitored in the switched plants and in the two unswitched control groups. Photosynthetic rates per unit leaf area recovered to the level of the 350 microliters per liter CO₂ grown control group within four to five days. To assess only nonstomatal limitations to photosynthesis, a measure of photosynthetic efficiencies was calculated (moles CO₂ fixed per square meter per second per mole intercellular CO₂). Photosynthetic efficiency also recovered to the levels of the 350 microliters per liter CO₂ grown controls within three to four days.

Recovery was correlated to a rapid depletion of the starch pool, indicating that the inhibition of photosynthesis is primarily a result of feedback inhibition. However, complete recovery may involve the repair of damage to the chloroplasts caused by excessive starch accumulation. The rapid and complete reversal of photosynthetic inhibition suggests that the appearance of large, strong sinks at certain developmental stages could result in reduction of the large starch accumulations and that photosynthetic rates could recover to near the theoretical capacity during periods of high photosynthate demand.

     

Characteristics of Five New Photoautotrophic Suspension Cultures Including Two Amaranthus Species and a Cotton Strain Growing on Ambient CO(2) Levels

Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO₂ (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO₂ levels (5%). Photoautotrophy was indicated by the requirement for CO₂ and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C₄Amaranthus species. The cells showed high dark respiration rates and had lower net CO₂ fixation under high O₂ conditions. Dark CO₂ fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO₂ conditions showed the unique properties of a high RuBPcase activation level in ambient CO₂ and a stable ability to show net CO₂ fixation in 21% O₂ conditions.     

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.     

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii

We have examined the induction of carbonic anhydrase activity in Chlamydomonas reinhardtii and have identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photoautotrophically on 5% CO₂, but its synthesis was induced under low concentrations of CO₂ (air levels of CO₂). In CW-15, a mutant of C. reinhardtii which lacks a cell wall, between 80 and 90% of the carbonic anhydrase activity of air-adapted cells was present in the growth medium. Furthermore, between 80 and 90% of the carbonic anhydrase is released if wild type cells are treated with autolysin, a hydrolytic enzyme responsible for cell wall degradation during mating of C. reinhardtii. These data extend the work of Kimpel, Togasaki, Miyachi (1983 Plant Cell Physiol 24: 255-259) and indicate that the bulk of the carbonic anhydrase is located either in the periplasmic space or is loosely bound to the algal cell wall. The polypeptide associated with carbonic anhydrase activity has a molecular weight of approximately 37,000. Several lines of evidence indicate that this polypeptide is responsible for carbonic anhydrase activity: (a) it appears following the transfer of C. reinhardtii from growth on 5% CO₂ to growth on air levels of CO₂, (b) it is located in the periplasmic space or associated with the cell wall, like the bulk of the carbonic anhydrase activity, (c) it binds dansylamide, an inhibitor of the enzyme which fluoresces upon illumination with ultraviolet light, (d) antibodies which inhibit carbonic anhydrase activity only cross-react with this 37,000 dalton species.     

C(4) Acid Metabolism and Dark CO(2) Fixation in a Submersed Aquatic Macrophyte (Hydrilla verticillata)

The CO₂ compensation point of the submersed aquatic macrophyte Hydrilla verticillata varied from high (above 50 microliters per liter) to low (10 to 25 microliters per liter) values, depending on the growth conditions. Plants from the lake in winter or after incubation in an 11 C/9-hour photoperiod had high values, whereas summer plants or those incubated in a 27 C/14-hour photoperiod had low values. The plants with low CO₂ compensation points exhibited dark ¹⁴CO₂ fixation rates that were up to 30% of the light fixation rates. This fixation reduced respiratory CO₂ loss, but did not result in a net uptake of CO₂ at night. The low compensation point plants also showed diurnal fluctuations in titratable acid, such as occur in Crassulacean acid metabolism plants. However, dark fixation and diurnal acid fluctuations were negligible in Hydrilla plants with high CO₂ compensation points.     

Effects of CO(2) Concentration on Rubisco Activity, Amount, and Photosynthesis in Soybean Leaves

Growth at an elevated CO₂ concentration resulted in an enhanced capacity for soybean (Glycine max L. Merr. cv Bragg) leaflet photosynthesis. Plants were grown from seed in outdoor controlled-environment chambers under natural solar irradiance. Photosynthetic rates, measured during the seed filling stage, were up to 150% greater with leaflets grown at 660 compared to 330 microliters of CO₂ per liter when measured across a range of intercellular CO₂ concentrations and irradiance. Soybean plants grown at elevated CO₂ concentrations had heavier pod weights per plant, 44% heavier with 660 compared to 330 microliters of CO₂ per liter grown plants, and also greater specific leaf weights. Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activity showed no response (mean activity of 96 micromoles of CO₂ per square meter per second expressed on a leaflet area basis) to short-term (~1 hour) exposures to a range of CO₂ concentrations (110-880 microliters per liter), nor was a response of activity (mean activity of 1.01 micromoles of CO₂ per minute per milligram of protein) to growth CO₂ concentration (160-990 microliters per liter) observed. The amount of rubisco protein was constant, as growth CO₂ concentration was varied, and averaged 55% of the total leaflet soluble protein. Although CO₂ is required for activation of rubisco, results indicated that within the range of CO₂ concentrations used (110-990 microliters per liter), rubisco activity in soybean leaflets, in the light, was not regulated by CO₂.     

Low bundle sheath carbonic anhydrase is apparently essential for effective c(4) pathway operation

Bundle sheath cells from leaves of a variety of C₄ species contained little or no carbonic anhydrase activity. The proportion of total leaf carbonic anhydrase in extracts of bundle sheath cells closely reflected the apparent mesophyll cell contamination of bundle sheath cell extracts as measured by the proportion of the mesophyll cell marker enzymes phosphoenolpyruvate carboxylase and pyruvate,Pi dikinase. Values of about 1% or less of the total leaf activity were obtained for all three enzymes. The recorded bundle sheath carbonic anhydrase activity was compared with a calculated upper limit of carbonic anhydrase activity that would still permit efficient functioning of the C₄ pathway; that is, a carbonic anhydrase level allowing a sufficiently high steady state [CO₂] to suppress photorespiration. Even before correcting for mesophyll cell contamination the activity in bundle sheath cell extracts was substantially less than the calculated upper limit of carbonic anhydrase activity consistent with effective C₄ function. The results accord with the notion that a deficiency of carbonic anhydrase in bundle sheath cells is vital for the efficient operation of the C₄ pathway.     

Mechanisms of inorganic carbon acquisition in two estuarine Rhodophyceans: Bostrychia scorpioides (Hudson) ex Kützing Montagne and Catenella caespitosa (Withering) L. M. Irvine

Marine macroalgae possess a range of mechanisms to increase the availability of CO₂ for fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase. Of these, possession of a periplasmic or external carbonic anhydrase and the ability to use bicarbonate ions is widely distributed. The mechanisms of carbon acquisition were studied in two estuarine red macroalgae Bostrychia scorpioides and Catenella caespitosa using a range of techniques. pH-drift and CO₂-depletion experiments at constant pH suggested that CO₂ is the main source of inorganic carbon in both species. Inhibitors indicated that internal and external carbonic anhydrase were present in both species. Inhibitors also suggested that uptake of bicarbonate is unlikely to be present (P < 0.05).     

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.     

Effects of High Atmospheric CO(2) and Sink Size on Rates of Photosynthesis of a Soybean Cultivar

The effect of sink strength on photosynthetic rates under conditions of long-term exposure to high CO₂ has been investigated in soybean. Soybean plants (Merr. cv. Fiskeby V) were grown in growth chambers containing 350 microliters CO₂ per liter air until pod set. At that time, plants were trimmed to three trifoliolate leaves and either 21 pods (high sink treatment) or 6 pods (low sink treatment). Trimmed plants were either left in 350 microliters CO₂ per liter of air or placed in 1000 microliters CO₂ per liter of air (high CO₂ treatment) until pod maturity. Whole plant net photosynthetic rates of all plants were measured twice weekly, both at 350 microliters CO₂ per liter of air and 1000 microliters CO₂ per liter of air. Plants were also harvested at this time for dry weight measurements. Photosynthetic rates of high sink plants at both measurement CO₂ concentrations were consistently higher than those of low sink plants, and those of plants given the 350 microliter CO₂ per liter of air treatment were higher at both measurement CO₂ concentrations than those of plants given the 1000 microliters CO₂ per liter of air treatment. When plants were measured under treatment CO₂ levels, however, rates were higher in 1,000 microliter plants than 350 microliter CO₂ plants. Dry weights of all plant parts were higher in the 1,000 microliters CO₂ per liter air treatment than in the 350 microliters CO₂ per liter air treatment, and were higher in the low sink than in the high sink treatments.     

Growth characteristics of hormone and vitamin independent photoautotrophic cell suspension cultures fromChenopodium rubrum

Summary This communication reports the photoautotrophic growth of hormone and vitamin independent cell suspension cultures ofChenopodium rubrum. The transfer of cells from stationary growth into fresh culture medium results in a high protein formation, followed by an exponential phase of cell division, whereas the onset of rapid chlorophyll formation is delayed for 4 days. At the stage of most rapid cell division there is no net synthesis of starch and sugar. When the cells enter stationary growth, there is a progressive accumulation of chlorophyll, sugar, and starch.Photoautotrophic cell cultures assimilate about 80–90 mol CO₂/mg chlorophyll X hour. Dark CO₂ fixation is about 3.7% to 2.2% of the light values during exponential and stationary growth, respectively. As shown by short-term¹⁴CO₂ fixation, CO₂ is predominantly assimilated through ribulosebisphosphate carboxylase via the Calvin pathway. There is a significant increase in the¹⁴C label of C₄ carboxylic acids in exponentially dividing cells as compared to cells from stationary growth. Thein vitro activity of phosphoenolpyruvate carboxylase and ribulosebisphosphate carboxylase is almost equal during exponential cell division. A decrease in cell division activity is accompanied by a significant change in the specific activities of both carboxylation enzymes. In non dividing cells from stationary growth the activity of ribulosebisphosphate carboxylase is greately enhanced and that of phosphoenolpyruvate carboxylase is reduced, documenting the development of carboxylation capacities typical for C₃-plants.The experimental results provide evidence that phosphoenolpyruvate carboxylase activity might be regulated by ammonia and could be involved in anaplerotic CO₂ fixation which supplies carbon skeletons of the citric acid cycle.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylene-diamine-tetraacetic acid - FDP fructose bisphosphate - F-6-P fructose-6-phosphate - G-6-P glucose-6-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PGA 3-phosphoglyceric acid - PEP phosphoenolpyruvate - RuDP ribulosebisphosphate