Chloroplast pH values and buffer capacities in darkened leaves as revealed by CO2 solubilization in vivo

Leaves of the C₃ plants Brassica oleracea L., Datura suaveolens Humb. & Bonpl. ex Willd., Helianthus annuus L. and Nicotiana tabacum L. with open stomata were exposed in a leaf chamber in the dark to CO₂ concentrations varying from 1 to 20% in air. When they were transferred back into CO₂-free air, CO₂ was rapidly released. It originated from dissolved CO₂ and from the bicarbonate in the chloroplast stroma, since vacuoles are acidic and chloroplasts contain carbonic anhydrase which rapidly liberates CO₂ from bicarbonate. The data were fitted to a model which accounts for the CO₂/bicarbonate equilibrium in buffers with different CO₂ concentrations and initial pH values. From this, pH values and CO₂-dependent pH changes in the chloroplast stroma were calculated. The full range of external CO₂ concentration caused acidic shifts up to 1 pH unit. The best fits of the data points were obtained with stromal buffer concentrations ranging from 45 to 65 mM and stromal pH values at low CO₂ between 7.5 and 7.9. Calculated buffer capacities ranged from 23 to 31 mM H⁺ per pH unit. The work shows that measurements of solubilized CO₂ are useful to investigate proton buffering and pH regulation in the chloroplast stroma of intact leaves.Abbreviation Chl chlorophyll This work was supported by Sonderforschungsbereich 251 of the University of Würzburg and the Volkswagenstiftung grant I-67762. We are very grateful to Dr. V. Oja for helpful advice.     

The role of carbon dioxide in the formation of end-products by Hymenolepis diminuta

The hypothesis that ambient CO₂ levels determine the end-products of energy metabolism excreted by Hymenolepis diminuta was tested by incubating the parasite in a range of CO₂ concentrations and measuring internal concentrations of adenine nucleotides and the excretion of organic acids. The strain of H. diminuta used was found to excrete mainly lactic acid and acetic acid. Succinic acid production was generally less than 5–10% of the total. At high CO₂ concentrations, the rate of excretion of lactic acid decreased while that of succinic acid increased, which conforms with the hypothesis. Acetic acid excretion did not vary significantly over the range of CO₂ concentrations used. Other results did not support the hypothesis. High CO₂ levels reduced the total amounts of acids excreted and the rate of succinic acid excretion was so small as to be ineffective in preventing the accumulation of H⁺ ions. When present in the incubation medium, succinic acid was taken up by H. diminuta. Lactic and acetic acid excretion was always sufficient to limit the accumulation of H⁺ ions. The conditions of incubation were shown not to be responsible for the low rates of succinic acid excreted. Incubation conditions and metabolic end-products were found to affect the rates of excretion of organic acids. There is thus a need, in work of this nature, to regulate and specify experimental conditions and to stipulate the strain of parasite used. The hypothesis was rejected and it was suggested that the energy metabolism of parasitic helminths is adapted to fluctuating O₂ and CO₂ tensions.     

CARBON‐USE STRATEGIES IN MACROALGAE: DIFFERENTIAL RESPONSES TO LOWERED PH AND IMPLICATIONS FOR OCEAN ACIDIFICATION1

Ocean acidification (OA) is a reduction in oceanic pH due to increased absorption of anthropogenically produced CO₂. This change alters the seawater concentrations of inorganic carbon species that are utilized by macroalgae for photosynthesis and calcification: CO₂ and HCO₃^? increase; CO₃^2? decreases. Two common methods of experimentally reducing seawater pH differentially alter other aspects of carbonate chemistry: the addition of CO₂ gas mimics changes predicted due to OA, while the addition of HCl results in a comparatively lower [HCO₃^?]. We measured the short‐term photosynthetic responses of five macroalgal species with various carbon‐use strategies in one of three seawater pH treatments: pH 7.5 lowered by bubbling CO₂ gas, pH 7.5 lowered by HCl, and ambient pH 7.9. There was no difference in photosynthetic rates between the CO₂, HCl, or pH 7.9 treatments for any of the species examined. However, the ability of macroalgae to raise the pH of the surrounding seawater through carbon uptake was greatest in the pH 7.5 treatments. Modeling of pH change due to carbon assimilation indicated that macroalgal species that could utilize HCO₃^? increased their use of CO₂ in the pH 7.5 treatments compared to pH 7.9 treatments. Species only capable of using CO₂ did so exclusively in all treatments. Although CO₂ is not likely to be limiting for photosynthesis for the macroalgal species examined, the diffusive uptake of CO₂ is less energetically expensive than active HCO₃^? uptake, and so HCO₃^?‐using macroalgae may benefit in future seawater with elevated CO₂.     

The effect of CO2 on potassium transport by Chlorella fusca

Abstract. The effect of CO₂ on net K⁺ uptake by Chlorella fusca grown on high CO₂ levels was examined by passing 1.5% CO₂ through algal suspensions gassed previously with air or CO₂-free air Addition of CO₂ in the light caused a large net uptake of K⁺ (initial velocity 4.2–9.2 mmol s^?1 m^?3 cells) which decreased the concentration of K⁺ in the supernatant from 0.1–0.2 mol m^?3 to 3–10 mmol m^?3. In the dark and in the presence of 30 mmol m^?3 DCMU, no effects were found. Measurement or the unidirectional K⁺ fluxes by using ⁸⁶Rb⁺ as a label showed that in the presence of 1.5% CO₂, influx of K⁺ was increased by a factor of 2–4 while efflux was inhibited completely. CO₂ hyperpolarized the membrane potential (determined through TPP⁺ uptake) from –120mV to –130 mV which could not explain the more than 15,000-fold K⁺ accumulations. In the light, CO₂ lowered the intracellular pH (determined with DMO) by 0.5 units. In the dark and in the presence of DCMU only, a small acidification of 0.1 units was found. During the first 15 min after addition of CO₂ the malate content of the cells increased from 0.7 to 1.5 mol m^?3 packed cells. On the basis of these and earlier results, CO₂-induced net K⁺ uptake is interpreted as a stimulation of an electroneutral ATP-dependent K⁺/H⁺ exchange at the plasmalemma. This exchange acts as a ‘pHstat’ by reducing the intracellular acidification caused by production of acidic assimilation products.     

THE INFLUENCE OF HYPOCAPNIA UPON INTRACELLULAR pH AND UPON SOME CARBOHYDRATE SUBSTRATES, AMINO ACIDS AND ORGANIC PHOSPHATES IN THE BRAIN

Abstract— In order to evaluate the influence of hypocapnia upon the energy metabolism of the brain, lightly anaesthetized rats were hyperventilated to arterial CO₂ tensions of 26, 15 and 10 mm Hg respectively, with subsequent measurements of intracellular pH and of tissue concentrations of carbohydrate substrates, amino acids and organic phosphates. At P_co1= 26 there was a moderate increase in the intracellular pH but when the P_co2 was reduced further to 10 mm Hg the intracellular pH returned to normal, or slightly subnormal, values. The reduction in P_Co2 was accompanied by increased cerebral cortical concentrations of lactate, pyruvate, citrate, α-ketoglutarate, malate and glutamate and by decreased aspartate concentrations. It is concluded that the accumulation of metabolic acids explains the normal value for intracellular pH at very low CO₂ tensions. Previous results obtained in man indicate that there is an increased anaerobic production of lactic acid in the brain in extreme hypocapnia. At comparable CO₂ tensions the present results showed a small fall in phosphocreatine and a small rise in ADP. However, since the ammonia concentrations were normal or decreased and since there was an increase in citrate, the results give no direct support to the hypothesis of an activation of phosphofructokinase. Since the cerebral venous P_o2 was reduced to 20 mm Hg at an arterial CO₂ tension of 10 mm Hg the accumulation of acids was probably secondary to tissue hypoxia. However, since there was no, or only a very small, increase in the calculated cytoplasmic NADH/NAD⁺ ratio, it appears less likely that acids accumulated due to lack of NAD⁺.     

The influence of enriched rhizosphere CO2 on N uptake and metabolism in wild-type and NR-deficient barley plants

Positive influences of high concentrations of dissolved inorganic carbon (DIC) in the growth medium of salinity-stressed plants are associated with carbon assimilation through phosphoenolpyruvate carboxylase (PEPc) activity in roots; and also in salinity-stressed tomato plants, enriched CO₂ in the rhizosphere increases NO^?₃uptake. In the present study, wild-type and nitrate reductase-deficient plants of barley (Hordeum vulgare L. cv. Steptoe) were used to determine whether the influence of enriched CO₂ on NO^?₃ uptake and metabolism is dependent on the activity of nitrate reductase (NR) in the plant. Plants grown in NH₄⁺and aerated with ambient air, were transferred to either NO₃^? or NH₄⁺ solutions and aerated with air containing between 0 and 6 500 μmol mol^?1 CO₂. Nitrogen uptake and tissue concentrations of NO₃^? and NH₄⁺ were measured as well as activities of NR and PEPc. The uptake of NO^?₃ by the wild-type was increased by increasing CO₂. This was associated with increased in vitro NR activity, but increased uptake of NO₃^? was found also in the NR-deficient genotype when exposed to high CO₂ concentrations; so that the influence of CO₂ on NO₃^? uptake was independent of the reduction of NO₃^? and assimilation into amino acids. The increase in uptake of NO₃^? in wild-type plants with enriched CO₂ was the same at pH 7 as at pH 5, indicating that the relative abundance of HCO₃^? or CO₂ in the medium did not influence NO₃^? uptake. Uptake of NH₄⁺ was decreased by enriched CO₂ in a pH (5 or 7) independent fashion. Thus NO₃^? and NH⁺₄ uptakes are influenced by the CO₂ component of DIC independently of anaplerotic carbon provision for amino acid synthesis, and CO₂ may directly affect the uptake of NO₃^? and NH₄⁺ in ways unrelated to the NR activity in the tissue.     

Concentrations of CO2 and O2 in floodwater and in internodal lacunae of floating rice growing at 1–2 metre water depths

Abstract. Environment and plant measurements were made to determine what factors may limit growth of deepwater and floating rice plants during partial or complete submergence. Field surveys included measurements of temperature, pH, light, O₂ and CO₂ in floodwater in Thailand. In addition, measurements were made of O₂ and CO₂ concentrations inside internodal lacunae of deepwater and floating rice growing at 0.5–2.0 m water depths. The bulk of measurements were taken during periods when the changes in water level were less than 50 mm d^?1. In the 0–0.02 m surface layer of floodwater at any location there were large changes in oxygen concentrations over diurnal cycles: there were decreases during the night down to 0.02–0.18 mol m^?3 O₂ at 0600 h and increases during the day to 0.13–0.28 mol m^?3 O₂ at 1500 h (0.28 mol m^?3 being 120% of the O₂ concentration of air saturated water at 30°C). During the day oxygen concentrations decreased with increasing water depth; concentrations just above the soil surface were occasionally zero. Most of this gradient disappeared during the night, and at dawn the 0.6 m surface layer of water had uniform low O₂ concentrations. O₂ concentrations were also measured during flash floods in Thailand. In contrast to the conditions with only small increases in water level, the O₂ concentrations in the water during flash floods were more uniform with depth and changed little over a diurnal cycle, the O₂ ranging between 0.14–0.19 mol m^?3. In most locations floodwater contained 0.2–1.9 mol m^?3 CO₂ and 0.7–1.6 mol m^?3 bicarbonate; however, in a location with acid sulphate soil CO₂ was only 0.05–0.2 mol m^?3, and bicarbonate concentrations were several fold lower. Concentrations of CO₂ in floodwater increased with increasing water depth. O₂ and CO₂ concentrations inside internodal lacunae of rice were determined in the field when water depth were 1–2 m. Concentrations of O₂ in internodes at the water surface were 16–20%, and decreased to 10% and 5% at 0.8 and 1.8 m water depth respectively. There was no diurnal cycle in O₂ concentrations inside internodes. In contrast, CO₂ concentrations in the lacunae increased with water depth and ranged from 1–3% in internodes at the water surface to 5–10% in internodes at 1.8 m water depth. There was evidence for a diurnal cycle in CO₂ concentrations in the basal internode near the soil surface, CO₂ increased during the day and decreased during the night. The above data are used to show that there is little or no relationship between gas concentrations in floodwater and internodal lacunae of rice plants. Results are discussed in relation to O₂ supply to submerged portions of rice and metabolism of these tissues at low O₂ concentrations.     

PHOTOSYNTHESIS AND CALCIFICATION BY EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) AS A FUNCTION OF INORGANIC CARBON SPECIES

To test the possibility of inorganic carbon limitation of the marine unicellular alga Emiliania huxleyi (Lohmann) Hay and Mohler, its carbon acquisition was measured as a function of the different chemical species of inorganic carbon present in the medium. Because these different species are interdependent and covary in any experiment in which the speciation is changed, a set of experiments was performed to produce a multidimensional carbon uptake scheme for photosynthesis and calcification. This scheme shows that CO₂ that is used for photosynthesis comes from two sources. The CO₂ in seawater supports a modest rate of photosynthesis. The HCO is the major substrate for photosynthesis by intracellular production of CO₂ (HCO+ H⁺→ CO₂+ H₂O → CH₂O + O₂). This use of HCO is possible because of the simultaneous calcification using a second HCO, which provides the required proton (HCO+ Ca²⁺→ CaCO₃+ H⁺). The HCO is the only substrate for calcification. By distinguishing the two sources of CO₂ used in photosynthesis, it was shown that E. huxleyi has a K_½ for external CO₂ of “only” 1.9 ± 0.5 μM (and a V_max of 2.4 ± 0.1 pmol·cell⁻¹·d⁻¹). Thus, in seawater that is in equilibrium with the atmosphere ([CO₂]= 14 μM, [HCO]= 1920 μM, at fCO₂= 360 μatm, pH = 8, T = 15° C), photosynthesis is 90% saturated with external CO₂. Under the same conditions, the rate of photosynthesis is doubled by the calcification route of CO₂ supply (from 2.1 to 4.5 pmol·cell⁻¹·d⁻¹). However, photosynthesis is not fully saturated, as calcification has a K_½ for HCO of 3256 ± 1402 μM and a V_max of 6.4 ± 1.8 pmol·cell⁻¹·d⁻¹. The H⁺ that is produced during calcification is used with an efficiency of 0.97 ± 0.08, leading to the conclusion that it is used intracellularly. A maximum efficiency of 0.88 can be expected, as NO uptake generates a H⁺ sink (OH⁻ source) for the cell. The success of E. huxleyi as a coccolithophorid may be related to the efficient coupling between H⁺ generation in calcification and CO₂ fixation in photosynthesis.     

Electrogenic proton secretion in the Hindgut of the desert locust,Schistocerca gregaria

Summary The cellular mechanisms responsible for rectal acidification in the desert locust, Schistocerca gregaria, were investigated in isolated recta mounted as flat sheets in modified Ussing chambers. Previous studies conducted in the nominal absence of exogenous CO₂ and HCO ₃ ^– suggested that the acidification was due to a proton-secretory rather than bicarbonate-reabsorptive mechanism (Thomson, R.B., Speight, J.D., Phillips, J.E. 1988. J. Insect Physiol. 34:829–837). This conclusion was confirmed in the present study by demonstrating that metabolic CO₂ could not contribute sufficient HCO ₃ ^– to the lumen to account for the rates of rectal acidification observed under the nominally CO₂/ HCO ₃ ^– -free conditions used in these investigations.Rates of luminal acidification (J _H ⁺) were completely unaffected by changes in contraluminal pH, but could be progressively reduced (and eventually abolished) by imposition of either transepithelial pH gradients (lumen acid) or transepithelial electrical gradients (lumen positive). Under short-circuit current conditions, the bulk of J _H ⁺ was not dependent on Na⁺, K⁺, Cl^–,Mg²⁺, or Ca²⁺ and was due to a primary electrogenic proton translocating mechanism located on the apical membrane. A small component (10–16%) of J _H ⁺ measured under these conditions could be attributed to an apical amiloride-inhibitable Na⁺/H⁺ exchange mechanism.This work was supported by operating grants to J.E.P. and postgraduate scholarships to R.B.T. from Natural Sciences & Engineering Research Council, Canada.     

Seasonal stability of sulfuric acid accumulation in the Dictyotales (Phaeophyceae)

Concerning the accumulation of S0₄^2‐ in cells, three types of species are known in the Dictyotales: (i) acidic type (high H₂S0₄ accumulation); (ii) high MgS0₄ accumulation type; and (iii) nonacidic type (low S0₄^2‐ accumulation). Seasonal changes of intracellular pH and concentrations of inorganic ions were examined in six dictyotalean species. In acidic species (Dictyopteris prolifera (Okamura) Okamura and Spatoglossum eras‐sum J. Tanaka), intracellular concentrations of S0₄^2‐ and H⁺ estimated by pH were high through all seasons. In high MgS0₄ accumulating species (Dictyotasp. and Padina arborescens Holmes), intracellular concentrations of S0₄^2‐ and Mg²⁺ were high through all seasons. In nonacidic species (Dictyota dichotoma (Hudson) Lamouroux and Dictyopteris undulata Holmes), intracellular concentration of sulfuric acid ion was low all year round.     

Evidence that inducible C4-type photosynthesis is a chloroplastic CO2-concentrating mechanism in Hydrilla, a submersed monocot

Hydrilla verticillata (L.f.) Royle exhibits an inducible C₄-type photosynthetic cycle, but lacks Kranz anatomy. Leaves in the C₄-type state (but not C₃-type) contained up to 5-fold higher internal dissolved inorganic carbon (DIC) concentrations than the medium, indicating that they possessed a CO₂-concentrating mechanism (CCM). Several lines of evidence indicated that the chloroplast was the likely site of CO₂ generation. From C₄-type leaf [DIC] measurements, the estimated chloroplastic free [CO₂] was 400 mmol m^?3. This gave a calculated 2% O₂ inhibition of photosynthesis, which was identical to the measured value, and provided independent evidence that the estimated [CO₂] was close to the true value. A homogeneous distribution of DIC in the C₄-type leaf could not account for such a high [CO₂], or the resultant low O₂ inhibition. For C₃-type leaves the estimated chloroplastic [CO₂] was only 7 mmol m^?3, which gave high, and similar, calculated and measured O₂ inhibition values of 22 and 26%, respectively. The CCM did not appear to be located at the plasma membrane, as it operated at low and high pH, indicating that it was independent of use of HCO₃^? from the medium. Also, both C₃^? and C₄-type Hydrilla leaves showed pH polarity in the light, with abaxial and adaxial boundary layer values of about pH 4·0 and 10·5, respectively. Thus, pH polarity was not a direct component of the CCM, though it probably improved access to HCO₃. Additionally, iodoacetamide and methyl viologen greatly reduced abaxial acidification, but not the steady-state CCM. Inhibitor studies suggested that the CCM required photosynthetically generated ATP, but Calvin cycle activity was not essential. Both leaf types accumulated DIC in the dark by an ATP-requiring process, possibly respiration, and C₄-type leaves fixed CO₂ at 11·8% of the light rate. The operation of a CCM to minimize photorespiration, and the ability to recapture respiratory CO₂ at night, would conserve DIC in a densely vegetated lake environment where daytime [CO₂] is severely limiting, while [O₂] and temperatures are high.     

Photosynthetic energy supply for NO3− assimilation in Scenedesmus

NO₃^?-dependent O₂ in synchronous Scenedesmus obtusiusculus Chod. in the absence of CO₂ is stoichiometric with NH₄⁺ excretion, indicating a close coupling of NO₃^? reduction to non-cyclic electron flow. Also in the presence of CO₂, NO₃^? stimulates O₂ evolution as manifested by an increase in the O₂/CO₂ ratio from 0.96 to 1.11. This quotient was increased to 1.36 by addition of NO₂^?, without competitive interaction with CO₂ fixation, indicating that the capacity for non-cyclic electron transport at saturating light is non-limiting for simultaneous reduction of NO₃^? and CO₂ at high rates. During incubation with NO₃^?+ CO₂, no NH₄⁺ is released to the outer medium, whereas during incubation with NO₂^?+ CO₂, excess NH₄⁺ is formed and excreted. NO₃^? uptake is stimulated by CO₂, and this stimulation is also significant when the cellular energy metabolism is restricted by moderate concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, whereas NO₃^? uptake in the absence of CO₂ is severely inhibited by the uncoupler. Also under energy-restricted conditions NO₃^? uptake is not competitive with CO₂ fixation. Antimycin A is inhibitory for NO₃^? uptake in the absence of CO₂, and there is no enhancement of NO₃^? uptake by CO₂ in the presence of antimycin A. It is assumed that the energy demand for NO₃^? uptake is met by energy fixed as triosephosphates in the Calvin cycle. Antimycin A possibly affects the transfer of reduced triose phosphates from the chloroplast to the cytoplasm. Active carbon metabolism also seems to exert a control effect on NO₃^? assimilation, inducing complete incorporation of all NO₃^? taken up into amino acids. This control effect is not functional when NO₂^? is the nitrogen source. Active carbon metabolism thus seems to be essential both for provision of energy for NO₃^? uptake and for regulation of the process.     

CO2 ACCUMULATION BY CHLORELLA SACCHAROPHILA (CHLOROPHYCEAE) AT LOW EXTERNAL pH: EVIDENCE FOR ACTIVE TRANSPORT OF INORGANIC CARBON AT THE CHLOROPLAST ENVELOPE1

The CO₂ uptake characteristics of the acid-tolerant alga Chlorella saccharophila (Krüger) Nadson have been examined. These, together with direct estimations of internal inorganic C concentrations, indicate that air-adapted cells of this alga possess a CO₂ accumulation mechanism similar to that reported in other members of the Chlorophyceae and in Cyanophyceae. This accumulation mechanism operates at external pH values of 4 and less. Under these conditions CO₂ must be the carbon species crossing the plasmlemma, and since preliminary evidence suggests that the transport mechanism is active primary uniport of HCO₃^–, it is proposed that the site of the CO₂ accumulation mechanism is the chloroplast envelope.     

Refixation of xylem sap CO2 in Populus deltoides

Vascular plants have respiring tissues which are perfused by the transpiration stream, allowing solubilization of respiratory CO₂ in the xylem sap. The transpiration stream could provide a conduit for the internal delivery of respiratory CO₂ to leaves. Trees have large amounts of respiring tissues in the root systems and stems, and may have elevated levels of CO₂ in the xylem sap which could be delivered to and refixed by the leaves. Xylem sap from the shoots of three Populus deltoides trees had mean dissolved inorganic carbon concentrations (CO₂+H₂CO₃+HCO^?₃) ranging from 0. 5 to 0. 9 mM. When excised leaves were allowed to transpire 1 mM[¹⁴C]NaHCO₃, 99. 6% of the label was fixed in the light. Seventy-seven percent of the label was fixed in major veins and the remainder was fixed in the minor veins. Autoradiography confirmed that label was confined to the vasculature. In the dark, approximately 80% of the transpired label escaped the leaf, the remainder was fixed in the major veins, slightly elevating dark respiration measurements. This indicates that the vascular tissue in P. deltoides leaves is supplied with a carbon source distinct from the atmospheric source fixed by interveinal lamina. However, the contribution of CO₂ delivered to the leaves in the transpiration stream and fixed in the veins was only 0. 5% of atmospheric CO₂ uptake. In the light 90% of the label was found in sugar, starch and protein, a pattern similar to that found for atmospheric uptake of[¹⁴C]CO₂. Compared with leaves labelled in the light, leaves labelled in the dark had more label in organic acid, amino acid and protein and less label in sugar and starch. After a 5-s pulse the majority of the label fed to petioles in both the light and the dark was found in malate. The majority of the label was found in malate at 120 s in the dark; only 2% of the label was found in phosphorylated compounds at 120 s. The proportion of label found in phosphorylated compounds increased from 17% at 5 s to 80% at 120 s in the light. This suggests that CO₂ delivered to leaves in the light via the transpiration stream is fixed in the veins, a small portion through dark fixation into malate, the remainder by C-3 photosynthesis.     

Soybean nodulation and N2 fixation response to drought under carbon dioxide enrichment

The combined effects of carbon dioxide (CO₂) enrichment and water deficits on nodulation and N₂ fixation were analysed in soybean [Glycine max (L.) Merr.]. Two short-term experiments were conducted in greenhouses with plants subjected to soil drying, while exposed to CO₂ atmospheres of either 360 or 700 μmol CO₂ mol^–1. Under drought-stressed conditions, elevated [CO₂] resulted in a delay in the decrease in N₂ fixation rates associated with drying of the soil used in these experiments. The elevated [CO₂] also allowed the plants under drought to sustain significant increases in nodule number and mass relative to those under ambient [CO₂]. The total non-structural carbohydrate (TNC) concentration was lower in the shoots of the plants exposed to drought; however, plants under elevated CO₂ had much higher TNC levels than those under ambient CO₂. For both [CO₂] treatments, drought stress induced a substantial accumulation of TNC in the nodules that paralleled N₂ fixation decline, which indicates that nodule activity under drought may not be carbon limited. Under drought stress, ureide concentration increased in all plant tissues. However, exposure to elevated [CO₂] resulted in substantially less drought-induced ureide accumulation in leaf and petiole tissues. A strong negative correlation was found between ureide accumulation and TNC levels in the leaves. This relationship, together with the large effect of elevated [CO₂] on the decrease of ureide accumulation in the leaves, indicated the importance of ureide breakdown in the response of N₂ fixation to drought and of feedback inhibition by ureides on nodule activity. It is concluded that an important effect of CO₂ enrichment on soybean under drought conditions is an enhancement of photoassimilation, an increased partitioning of carbon to nodules and a decrease of leaf ureide levels, which is associated with sustained nodule growth and N₂ rates under soil water deficits. We suggest that future [CO₂] increases are likely to benefit soybean production by increasing the drought tolerance of N₂ fixation.     

Cyclic GMP Inhibits a Pharmacologically Distinct Na+/H+ Exchanger Variant in Cultured Rat Astrocytes via an Extracellular Site of Action

Abstract: There is growing evidence that cyclic GMP (cGMP) plays important roles in the brain. In cultured rat astrocytes, we observed that the cGMP-inducing C-type natriuretic peptide (CNP) and cGMP analogues caused a decrease in intracellular pH (pH_i). To examine whether this effect was due to inhibition of an Na⁺/H⁺ exchanger (NHE), we acidified cells by replacing extracellular Na⁺ by choline and examined the kinetics of the pH_i recovery that occurred on reintroduction of Na⁺ in the extracellular medium. Both CNP and amiloride analogues inhibited the Na⁺-dependent pH_i recovery, even in the nominal absence of CO₂/HCO₃^?. This indicated that CNP inhibited the activity of an exchanger that was Na⁺-dependent, HCO₃^?-independent, and sensitive to known inhibitors of NHE. However, comparison of the potencies of four distinct amiloride analogues revealed a pharmacological profile that was different from that of any other NHE characterized to date. cGMP mimicked the effect of CNP on sodium-dependent pH_i recovery, but the native nucleotide was as potent as membrane-permeant analogues. Intracellularly produced cGMP was very rapidly exported out of astrocytes. Probenecid and niflumic acid slowed down the rate of cGMP egression and inhibited the effect of CNP on Na⁺-dependent recovery, but not that of extracellular cGMP. Altogether, our data indicate that cGMP inhibits a novel type of NHE in astrocytes via an extracellular site of action. If these results with primary cultures transfer to brain, this phenomenon may constitute a mechanism by which natriuretic peptides exert some of their actions in the brain, as pH_i transients have been shown to modulate several important astrocytic functions.     

Acute toxicity of carbon dioxide to juvenile marine shrimp Litopenaeus vannamei (Boone 1931)

Elevated concentrations of dissolved carbon dioxide (CO₂) and reduced pH levels are observed during the culture and transportation of aquatic organisms. Studies on the toxicity effects of CO₂ in penaeid shrimp are scarce when compared to the amount of research in fish. The objective of the present study was to determine the lethal concentration and safety levels of CO₂ for juvenile white shrimp Litopenaeus vannamei. Juveniles (1.76 ± 0.36 g) were exposed for 96 h to one of six concentrations of dissolved CO₂ (14.5, 23.8, 59.0, 88.0, 115.0, and 175.0 mg/L) or a control condition (without the addition of CO₂), and their survival was monitored for 96 h. The LC₅₀ values with 95% confidence limits at 24, 48, 72, and 96 h were 130.05 (104.2–162.1), 77.2 (73.8–80.02), 69.65 (65.47–74.32), and 59.12 (53.08–66.07) mg/L of CO₂, respectively. The calculated safety level was 5.9 mg/L of CO₂, and the highest concentration that did not induce significantly higher mortality than that observed in controls (NOEC) was 23.8 mg/L of CO₂. We recommend that CO₂ levels should be kept below the safety level obtained in this study.     

CO2 uptake and transport in leaf mesophyll cells

Abstract The acquisition of inorganic carbon for photosynthetic assimilation by leaf mesophyll cells and chloroplasts is discussed with particular reference to membrane permeation of CO₂ and HCO^?₃. Experimental evidence indicates that at the apoplast pH normally experienced by leaf mesophyll cells (pH 6–7) CO₂ is the principal species of inorganic carbon taken up. Uptake of HCO^?₃ may also occur under certain circumstances (i.e. pH 8.5), but its contribution to the net flux of inorganic carbon is small and HCO^?₃ uptake does not function as a CO₂-concentrating mechanism. Similarly, CO₂ rather than HCO^?₃ appears to be the species of inorganic carbon which permeates the chloroplast envelope. In contrast to many C₃ aquatic plants and C₄ plants, C₃ terrestrial plants lack specialized mechanisms for the acquisition and transport of inorganic carbon from the intercellular environment to the site of photosynthetic carboxylation, but rely upon the diffusive uptake of CO₂.     

INTERACTIONS BETWEEN LIGHT,NH4+, AND CO2 IN BUOYANCY REGULATION OF ANABAENA FLOS-AQUAE (CYANOPHYCEAE)1

Buoyancy of the gas-vacuolate alga Anabaena flosaquae Brébisson was measured under various levels of light, NH₄⁺, and CO₂. At high irradiance (50 μE · m^?2·^?1) the alga was non-buoyant regardless of the availability of CO₂ and NH₄⁺. At low irradiance (≤10 μE · m ^?2· s^?1) buoyancy was controlled by the availability of NH₄⁺ and CO₂. When NH₄⁺ was abundant, algal buoyancy was high over a wide range of CO₂ concentrations. In the absence of NH₄⁺, algal buoyancy was reduced at high CO₂ concentrations, however as the CO₂ concentration declined below about 5 μmol · L^?1, algal buoyancy increased. These results help explain why gas vacuolate, nitrogen-fixing blue-green algae often form surface blooms in eutrophic lakes.     

Effects of elevated CO2 on the protein concentration of food crops: a meta-analysis

Meta‐analysis techniques were used to examine the effect of elevated atmospheric carbon dioxide [CO₂] on the protein concentrations of major food crops, incorporating 228 experimental observations on barley, rice, wheat, soybean and potato. Each crop had lower protein concentrations when grown at elevated (540–958 μmol mol^?1) compared with ambient (315–400 μmol mol^?1) CO₂. For wheat, barley and rice, the reduction in grain protein concentration was ～10–15% of the value at ambient CO₂. For potato, the reduction in tuber protein concentration was 14%. For soybean, there was a much smaller, although statistically significant reduction of protein concentration of 1.4%. The magnitude of the CO₂ effect on wheat grains was smaller under high soil N conditions than under low soil N. Protein concentrations in potato tubers were reduced more for plants grown at high than at low concentrations of ozone. For soybean, the ozone effect was the reverse, as elevated CO₂ increased the protein concentration of soybean grown at high ozone concentrations. The magnitude of the CO₂ effect also varied depending on experimental methodology. For both wheat and soybean, studies performed in open‐top chambers produced a larger CO₂ effect than those performed using other types of experimental facilities. There was also indication of a possible pot artifact as, for both wheat and soybean, studies performed in open‐top chambers showed a significantly greater CO₂ effect when plants were rooted in pots rather than in the ground. Studies on wheat also showed a greater CO₂ effect when protein concentration was measured in whole grains rather than flour. While the magnitude of the effect of elevated CO₂ varied depending on the experimental procedures, a reduction in protein concentration was consistently found for most crops. These findings suggest that the increasing CO₂ concentrations of the 21st century are likely to decrease the protein concentration of many human plant foods.