Regulation of Catalase Activity in Leaves of Nicotiana sylvestris by High CO(2)

The effect of high CO₂ (1% CO₂/21% O₂) on the activity of specific forms of catalase (CAT-1, -2, and -3) (EA Havir, NA McHale [1987] Plant Physiol 84: 450-455) in seedling leaves of tobacco (Nicotiana sylvestris, Nicotlana tabacum) was examined. In high CO₂, total catalase activity decreased by 50% in the first 2 days, followed by a more gradual decline in the next 4 days. The loss of total activity resulted primarily from a decrease in CAT-1 catalase. In contrast, the activity of CAT-3 catalase, a form with enhanced peroxidatic activity, increased 3-fold in high CO₂ relative to air controls after 4 days. Short-term exposure to high CO₂ indicated that the 50% loss of total activity occurs in the first 12 hours. Catalase levels increased to normal within 12 hours after seedlings were returned to air. When seedlings were transferred to air after prolonged exposure to high CO₂ (13 days), the levels of CAT-1 catalase were partially restored while CAT-3 remained at its elevated level. Levels of superoxide dismutase activity and those of several peroxisomal enzymes were not affected by high CO₂. Total catalase levels did not decline when seedlings were exposed to atmospheres of 0.04% CO₂/5% O₂ or 0.04% CO₂/1% O₂, indicating that regulation of catalase in high CO₂ is not related directly to suppression of photorespiration. Antibodies prepared against CAT-1 catalase from N. tabacum reacted strongly against CAT-1 catalase from both N. sylvestris and N. tabacum but not against CAT-3 catalase from either species. This observation, along with the rapid changes in CAT-1 and the much slower changes in CAT-3 suggest that one form is not directly derived from the other.     

Alteration of collagen synthesis in lung organ cultures by hyperoxic environments

Organ cultures of neo-natal rat lungs continue to accumulate collagen at a nearly linear rate for 5 days, when maintained in an atmosphere consisting of 5% CO₂ in air. Nearly 70% of the collagen synthesized in these cultures is Type I and 30%, Type III. When these cultures are maintained in 5% CO₂ + 95% O₂, the per cell accumulation of collagen increased. Although there is no significant change in the amount of radiolabeled proline incorporated, the rate of incorporation per cell is increased since there appear to be fewer cells in the O₂-treated cultures. These parameters of collagen synthesis parallel lung O₂ injury in vivo. There is a shift in the type of collagen synthesized, nearly 70% being Type III and 30%, Type I in O₂-exposed cultures. This finding is reminiscent of tissue repair after injury. Such changes in collagen may contribute to pulmonary connective tissue disorders in oxidant-induced lung injury.     

Glucose and lactic acid trends in suspension cultures of two established mammalian cell strains in chemically defined media

In replicated 30 to 40-ml suspension cultures of rapidly proliferating monkey kidney cells of a comparatively fragile strain, the rates of glucose utilization and lactic acid accumulation averaged about 400 micrograms and 110 micrograms per 10⁶ cells per day respectively, with average molar La/Gl ratios of 0.48. These two rates of glucose utilization and lactic acid accumulation were about 4 × and 10 × as high as the corresponding rates in comparable cultures of the hardier strain 2071-L mouse fibroblasts under the same conditions, with average molar La/Gl ratios of 0.16. In comparable but nonproliferating suspension cultures of the same strain of monkey kidney cells, during about 3 weeks the rates were extremely high, with about 710 micrograms glucose utilized and 445 micrograms lactic acid accumulated per 10⁶ cells per day, with average molar La/Gl ratios of 1.37. The rates of glucose uptake and lactic acid accumulation were higher in the nonproliferating cultures aerated with 5% CO₂ in air than in those aerated with 10% CO₂ in air. This difference was associated with pH, which was higher in the former group. It was concluded that with this fragile strain of monkey Kidney cells(1) in nonproliferating cultures the cells were metabolizing actively but with a marked tendency to higher La/Gl ratios, (2) in the proliferating cultures the high rates of glucose utilization and lactic acid accumulation were definitely not directly correlated with the rate of growth, and (3) in none of the cultures was the amount of glucose remaining in the fluid at fluid changes so low as to have been a limiting factor. Information in the literature concering glucose utilization and lactic acid production by cells vitro is voluminous and in some respects contradictory. In the present study the rates were unexpectedly high for the monkey kidney cells, particularly those in the otherwise apparently inactive nonproliferating cultures. The data seem to be unique, in that an established strain of cells in chemically defined medium in suspension cultures has been characterized for these metabolic parameters in both proliferating cultures and in equivalent nonproliferating cultures under directly comparable conditions. The concept was developed that since these monkey kidney cells are obviously more fragile than the other cells examined, the complex physical stresses imposed upon these cells in agitated cultures can be modified and lessened in order to permit growth. Lessening of such mechanical stress waa brought about in several ways, of which only the smaller flask size seemed to be at least partly effective. Increasing either the concentration or the viscosity type of Methocel waa not effective.     

DECREASED RATE OF GLUCOSE UTILIZATION BY RAT BRAIN IN VIVO AFTER EXPOSURE TO ATMOSPHERES CONTAINING HIGH CONCENTRATIONS OF CO2

—(1) The effects of exposure of rats to increased atmospheric concentrations of CO₂ on brain metabolism in vivo were studied. (2) After 2·5 min exposure to an atmosphere of 20% CO₂, the rate of glucose utilization by brain decreased from 0·61 μmol/min per g to 0·32 μmol/min per g and remained between 0·3 and 0·4 μmol/min per g for 60 min, the longest interval studied. O₂ utilization, calculated from the arteriovenous difference of O₂ across the brain and blood flow, was 3·5 μmol/min per g in controls and was 4·7 μmol/min per g after 5 min in the 20% CO₂ atmosphere. (3) The concentrations of glucose, glucose 6-phosphate and aspartate were increased during the first 10 min of CO₂ exposure whereas the concentrations of other glycolytic intermediates, tricarboxylic acid cycle intermediates and glutamate were decreased. The amount of endogenous substrate which disappeared during the first 10 min was sufficient, if used to supplement glucose as a fuel, to maintain the O₂ consumption at, or slightly above, the control level. Glutamate and lactate were quantitatively the most important energy sources. (4) The mechanism whereby‘CO₂ decreased the rate of glucose utilization is uncertain. The initial rise in glucose 6-phosphate and fall in fructose 1,6-diphosphate concentrations suggested that an inhibition of phosphofructokinase was responsible. However, after 60 min in 20% CO₂, the concentrations of both of these metabolites returned to normal while the rate of glucose utilization remained depressed.     

Storage of Shredded Cabbage under a Dynamically Controlled Atmosphere of High O2 and High CO2

Shredded cabbage (Brassica oleracea L., Capitata group) was stored under a dynamically controlled atmosphere (DCA) and modified atmosphere (MA) at 5°C. Quality factors were measured every 2 days. Browning was suppressed as the CO₂ concentration was increased (0% to 15%), with no influence from O₂ concentration (2.5% to 10%). The development of an off-odor was markedly delayed with an increase in O₂ concentration, such an odor being detected after 6 days at 2.5% O₂,8 to 10 days at 5% to 7.5% O₂, and not at all above 10 days at 10% O₂, while the off-odor development was little affected by CO₂ concentration (5% to 15%). Total sugar, polyphenolics, total ascorbate, and total microbial count were little influenced by O₂ and CO₂. These results show that shredded cabbage can be kept in good condition with a combined high O₂ and high CO₂ atmosphere. These findings are largely different from those for MA storage.     

Effect of oxygen on photosynthesis by spinach leaf protoplasts

The photosynthetic CO₂ fixation by spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts was inhibited by substituting an atmosphere of N₂ with one of either air (21% O₂) or 100% O₂. The inhibitory effect of 100% O₂ was greater than that of air. The mode of inhibition by 100% O₂ and air was competitive with respect to CO₂; Ki(O₂) value was 0.32 mM at pH 7 and 0.28 mM at pH 8.5 The labeling patterns of compounds in protoplasts exposed to ¹⁴CO₂ in light after transferring them from N₂ to O₂ atmospheres were examined. There was no detectable ¹⁴CO₂ incorporation into glycolate under anaerobic and O₂ atmospheres; a more marked labeling of glycine occurred under an oxidative environment compared to that under the anaerobic condition, presumably because of a rapid transformation of glycolate to glycine in the protoplasts.     

Effects of Acetazolamide (Diamox), Ethoxzolamide and High Levels of CO2 on Carbonic Anhydrase, Photosystem Activity, and Oxygen Evolution in Euglena gracilis

Investigations using steady-state culture conditions indicate that carbonic anhydrase activity is correlated to the photosynthetic rate in Euglena in some but not all circumstances. When cultures grown with 5% CO₂ were changed to air growth, the photosynthetic rate was independent of the carbonic anhydrase activity. While experiments using the inhibitor acetazolamide indicated a close correlation between photosynthetic capacity and carbonic anhydrase activity, the inhibitor was found to be nonspecific. Acetazolamide altered photosystem activities directly as measured by the photoreduction of DCPIP in chloroplast preparations, whole-cell fluorescence transients of chlorophyll a, and by whole chain photoelectron flow. Ethoxzolamide, another inhibitor of carbonic anhydrase, was also found to inhibit photosystem activities, i.e., the photoreduction of DCPIP, and in vivo photoelectron flow, at high concentrations. Cells grown in 5% CO₂ were less sensitive to the effects of acetazolamide than cells exposed to air. The rate of electron flow in chloroplasts from cells grown with 5% CO₂ and exposed to 10 mM acetazolamide was 2.5-fold faster than that of chloroplasts from air-grown cells exposed to the same concentration of inhibitor. The whole cell chlorophyll a fluorescence transients of cultures grown with high CO₂ were completely different from those of air-grown cells and also showed fewer effects on exposure to acetazolamide. These results suggest a reevaluation of the hypothesis that carbonic anhydrase activity regulates photosynthesis. It is also apparent that results from air-grown and 5% CO₂-grown cultures cannot be directly compared in such studies.     

Adaptation of the Cyanobacterium Anabaena variabilis to Low CO(2) Concentration in Their Environment

The rate of adaptation of high CO₂ (5% v/v CO₂ in air)-grown Anabaena to a low level of CO₂ (0.05% v/v in air) was determined as a function of O₂ concentration. Exposure of cells to low (2.6%) O₂ concentration resulted in an extended lag in the adaptation to low CO₂ concentration. The rate of adaptation following the lag was not affected by the concentration of O₂. The length of the lag period is markedly affected by the O₂/CO₂ concentration ratio, indicating that the signal for adaptation to low CO₂ may be related to the relative rate of ribulose-1,5-bisphosphate carboxylase/oxygenase activities, rather than to CO₂ concentration proper. This suggestion is supported by the observed accumulation of phosphoglycolate following transfer of cells from high to low CO₂ concentration.     

Ischemic myocardial injury in cultured heart cells: Leakage of cytoplasmic enzymes from injured cells

Summary An in vitro model of myocardial ischemia has been established with primary monolayer cultures of postnatal rat myocardial cells. Ischemic conditions were simulated in vitro by subjecting the myocardial cell cultures to various levels of oxygen and glucose deprivation. The experimental protocol consisted of treatment with 20% or 0% O₂ and 1000, 500 or 0 mg glucose per 1 of medium for 4 or 24 hr. Control cultures were treated with 20% O₂ and 1000 mg glucose. After the ischemic treatments, cultures of beating muscle (M) cells were evaluated for signs of injury, i.e. leakage of cytoplasmic enzymes into the culture medium. Differences were found in leakage of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) from the cultures that were exposed to partial ischemia of glucose deprivation and from those cultures that were exposed to total ischemia of oxygen and glucose deprivation. Glucose deprivation alone resulted in a slight-to-moderate loss of LDH and CPK from the cells, whereas total ischemia resulted in a significant release of the two cytoplasmic enzymes. When the cultures were allowed to recover after ischemic treatment in complete medium (1000 mg glucose) and a normal atmosphere of 20% O₂, they had levels of LDH leakage comparable to those of control cultures. Cell viability and total protein content of the ischemic cultures did not differ significantly from controls. This study was supported by Research Grant HL 18647 from the National Heart, Lung, and Blood Institute.     

OBSERVATIONS ON CELL DEVELOPMENT IN CHLAMYDOMONAS SEGNIS (CHLOROPHYCEAE) AT LOW AND HIGH CARBON DIOXIDE TENSION1

Some cell cycle events were compared in Chlamydomonas segnis Ettl during its development in synchronous cultures (12:12 LD) supplied with air and air enriched with 5% CO₂. In cultures bubbled with air, growth resulted in production of 2 relatively small zoospores. In cultures provided with 5% CO₂, 4 large zoospores were formed but not released in darkness unless the cultures were bubbled with CO₂-free air and/or exposed to light. Respiration in zoospores was inhibited by high CO₂ tension. In cultures maintained under continuous illumination for one cell cycle, provision of 5% CO₂ led to enhanced growth, a relatively long S-phase and a 4 h delay of the second cell division. In such cultures, the DNA content of parental cells (12 h L) was insufficient to support two cell divisions. The RNA/DNA ratio of the resulting zoospores was 10 compared to 4 in air cultures. These results provided evidence that the delay of the second cell division was a consequence of the delay in DNA production.     

Some biophysical principles underlying the controlled atmosphere storage of plant material

Controlled atmosphere (CA) storage is of use for commodities which potentially can undergo rapid and unacceptable biochemical change. In air, the oxygen status of most plant material, including fleshy storage organs and fruits, suffices, even in the centre, for cytochrome oxidase to be fully saturated. Conflict of evidence exists as to possible O₂ and CO₂ gradients in fruit which, though physiologically unimportant in air, could be important under CA conditions. CA storage gives possible control of internal O₂ from o to about 80–95%; internal CO₂ from about 3–4 to 100%; and both simultaneously to intermediate values. Calculated molarities of dissolved O₂, CO₂ and ethylene are given for various atmospheres. The differences in the O₂ concentrations recommended for different varieties of apple are not readily explicable. Varietal differences in susceptibility to CO₂ injury could possibly result from anatomical, rather than biochemical, differences. This could be determined partly by resolving the conflict of evidence mentioned above. Variability of plant material prevents precise control of intercellular atmosphere; recommended atmospheres can be designed only to avoid completely anaerobic conditions and a harmful level of CO₂ in the centre of the least permeable individual fruit or vegetable. Effects of low O₂ and high CO₂ are briefly described.     

Effect of CO2, CO2 and Diamox on photosynthesis and photorespiration in Chlamydomonas reinhardtii (green alga) and Anacystis nidulans (Cyanobacterium, blue-green alga)

Photorespiration by Chlamydomonas reinhardtii and Anacystis nidulans was measured as the oxygen inhibition of CO₂ uptake and the CO₂ compensation points. Net photosynthesis was oxygen dependent in Chlamydomonas grown in 5% CO₂, but CO₂ insensitive in cultures bubbled with air. Anacystis, even when cultured in 5% CO₂, exhibited an CO₂ insensitive net photosynthesis. The CO₂ compensation point of Chlamydomonas grown in cultures bubbled with air and Anacystis grown in 5% CO₂ enriched air, were reached shortly after the measurement was begun and the values were very low, less than 10 μl CO₂ 1^?1; while Chlamydomonas grown in 5% CO₂ enriched air for 4 days showed a high, but temporary CO₂ compensation point (60 μl CO₂ 1^?1). After a two hour adaptation in low CO₂, a stable, low CO₂ compensation point was reached. It seems that photorespiration can only be detected by the methods used in this study when the algae are cultured in high CO₂, but a mechanism exists which blocks photorespiration when the green algae are adapted to low CO₂ concentrations. When Chlamydomonas was treated with Diamox, an inhibitor of carbonic anhydrase, after cultivation in low CO₂ (air), the cells behaved as if they had been grown in high CO₂. They showed an oxygen sensitive net photosynthesis and a high CO₂ compensation point. This indicates that carbonic anhydrase plays an important role in the regulation of a measurable photorespiration in Chlamydomonas. The results are discussed in relation to previous observations of photorespiration measured by enzyme assay, metabolic products and gas exchange properties.     

Control of the circadian rhythm of carbon dioxide assimilation in Bryophyllum leaves by exposure to darkness and high carbon dioxide concentrations

The circadian rhythm of CO₂ assimilation in detached leaves of Bryophyllum fedtschenkoi at 15° C in normal air and continuous illumination is inhibited both by exposure to darkness, and to an atmosphere enriched with 5% CO₂. During such exposures substantial fixation of CO₂ takes place, and the malate concentration in the cell sap increases from about 20 mM to a constant value of 40–50 mM after 16 h. On transferring the darkened leaves to light, and those exposed to 5% CO₂ to normal air, a circadian rhythm of CO₂ assimilation begins again. The phase of this rhythm is determined by the time the transfer is made since the first peak occurs about 24 h afterwards. This finding indicates that the circadian oscillator is driven to, and held at, an identical, fixed phase point in its cycle after 16 h exposure to darkness or to 5% CO₂, and it is from this phase point that oscillation begins after the inhibiting condition is removed. This fixed phase point is characterised by the leaves having acquired a high malate content. The rhythm therefore begins with a period of malate decarboxylation which lasts for about 8 h, during which time the malate content of the leaf cells must be reduced to a value that allows phosphoenolpyruvate carboxylase to become active. Inhibition of the rhythm in darkness, and on exposure to 5% CO₂ in continuous illumination, appears to be due to the presence of a high concentration of CO₂ within the leaf inhibiting malic enzyme which leads to the accumulation of high concentrations of malate in the leaf cells. The malate then allosterically inhibits phosphoenolpyruvate carboxylase upon which the rhythm depends. The results give support to the view that malate synthesis and breakdown form an integral part of the circadian oscillator in this tissue.Abbreviations B. Bryophyllum - PEPCase phosphoenolpyruvate carboxylase     

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.     

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.     

Glycolate Formation and Excretion by Chlorella pyrenoidosa and Netrium digitus

Conditions are described whereby suspensions of Chlorella pyrenoidosa and Netrium digitus photosynthetically biosynthesize and excrete glycolate continuously in high yields. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate-linked enzymes, increased the excretion of glycolate approximately 4-fold in 1 hour (8 millimolar) and 20-fold in 4 hours (40 millimolar) in the presence of 0.2% CO₂ in air. The amount of glycolate excreted in the presence of aminooxyacetate and an atmosphere of 0.2% CO₂ in air equaled or exceeded the amount excreted in 0.2% CO₂ in O₂ minus aminooxyacetate. CO₂ and light were required for glycolate excretion. Aminooxyacetate also stimulated photosynthetic glycolate excretion in an atmosphere of 0.2% CO₂ in nitrogen or helium, although the stimulation was not as great as when air or O₂ was present.

The excreted glycolate was converted to H₂ and CO₂ by the combined action of glycolic oxidase and the formic hydrogenlyase complex found in Escherichia coli in total conversion yields of 80%.

     

Partial reversal by sodium ascorbate of hyperoxia-induced damage to HEp-2 cell cultures

Summary Hyperoxia induced cellular damage was used as an experimental model system for examining the ameliorative role of antioxidants. Multiplication of HEp-2 cells in monolayer culture was inhibited after exposure to 100% O₂ either hyperbarically at 3 atm absolute (atma) or normobarically at 1 atma for periods from 15 s to 4 h. The inhibition was characterized by a slower rate of replication for a period from 1 to 3 d after exposure than in unexposed cultures, and then massive cellular death. Less killing followed exposure to normobaric O₂ than to hyperbaric O₂, and the shorter the period of exposure to hyperoxia the less killing. Addition of 100 μg/ml of sodiuml-ascorbate to unexposed cultures enhanced growth (cell number at 6 d) almost twofold. When added ascorbate was present only during hyperoxic exposure (but not afterward), subsequent growth in air was enhanced 1.6-fold. However, when cells were exposed without added ascorbate, there was from 2 to 12-fold greater growth in air in the presence of the added ascorbate (as compared to exposed controls). This greater growth was always only a partial reversal of the lethal effect resulting from hyperoxia. Addition of 25 μg/ml catalase did not affect control or exposed cultures. Addition of ascorbate plus catalase was not as effective as ascorbate alone in promoting growth; the catalase moiety antagonized some of the growth enhancing influence of ascorbate. This suggests that extracellular H₂O₂ was not a factor in the lethal effect resulting from hyperoxia.     

Aphid response to elevated ozone and CO2

Numerous reports indicate that pollution stress caused by sulphur dioxide (SO₂), oxies of nitrogen or fluorides promote aphid growth on herbaceous and woody plants. At SO₂ exposures, the response curve of aphids is bell-shaped having the peak at 100 ppb. This curvilinear response is related to physiological stress responses of host plants exposed to pollutants. On the other hand, observations of aphid performance on ozone-exposed (O₃) or elevated carbon dioxide-exposed (CO₂) plants have given very variable results. Depending on the duration and concentration of O₃ or elevated CO₂ exposure or the age of the exposed plants, aphid growth on the same plants either decreased or increased in comparison to growth on control plants grown in filtered air. The results of these studies suggest that there is no general air pollution-induced plant stress that triggers aphid outbreaks on plants. Plants grown in elevated CO₂ usually have higher C/N ratios than plants grown in current ambient CO₂ atmosphere. A reduced proportion of nitrogen in the plant foliage decreases growth of chewing herbivorous insects, but the few studies of elevated CO₂ effects on sucking insects such as aphids have not yielded similar consistent effects. The present paper reviews recent studies of elevated CO₂ effects on aphids and discusses the effects of combined elevated O₃ and CO₂ exposures on aphid performance on woody plants using pine and birch aphids as examples.     

INDUCIBLE MECHANISMS FOR HCO3– UTILIZATION AND REPRESSION OF PHOTORESPIRATION IN PROTOPLASTS AND THALLI OF THREE SPECIES OF ULVA (CHLOROPHYTA)1

Thalli of Ulva reticulata Forskaal, Ulva rigida C. Ag., and Ulva pulchra Jaasund were incubated at different concentrations of dissolved CO₂. Incubation at a high CO₂ concentration resulted in decreased oxygen evolution rate and lower affinity for inorganic carbon at high pH conditions, i.e. the ability to use HCO₃^– as a carbon source was reduced. This effect was reversible, and plants regained this HCO₃^– uptake capacity when transferred to air concentrations of CO₂. The phytosynthetic oxygen evolution rate of plants grown at high CO₂ concentration was reduced by high O₂ concentrations, whereas thalli and protoplasts from cultures grown at air concentration were not affected. This is interpreted as a deactivation of the carbon-concentrating mechanism during conditions of high CO₂ resulting in high photorespiration when plants are exposed to high O₂ concentrations. Protoplasts were not affected by high O₂ to the same extent and were not able to utilize HCO₃^– from the medium. The algae were able to grow at very low CO₂ concentrations, but growth was suppressed when an inhibitor of external carbonic anhydrase was present. Assay of carbonic anhydrase activities showed that external and internal CA activities were lower in plants grown at a high CO₂ concentration compared to plants grown at a low concentration of CO₂. Possible mechanisms for HCO₃^– utilization in these Ulva species are discussed.     

Effects of doubled atmospheric CO2 concentration on the growth and photosynthesis of Chlamydomonas reinhardtii (Volvocales, Chlorophyceae)

The freshwater microalga, Chlamydomonas reinhardtii Dangeard, was cultured under 350 and 700 ppmv CO₂ to determine the impact of doubled atmospheric CO₂ concentration on its growth and photosynthesis. No significant difference was observed in the specific growth rate, photosynthetic efficiency, maximal net photo‐synthetic rate and light‐saturating point between the low and high CO₂ cultures. Both the low‐ and high‐CO₂‐grown cells showed reduced light‐dependent O₂ evolution rate and photochemical efficiency (F_v/F_m) owing to photoinhibition when exposed to high photon flux density. However, high‐CO₂‐grown cells were less photoinhibited, and showed better recovery in dim light or darkness during the initial period of the recovery process.