EXTRACTION,PARTIAL PURIFICATION AND CHARACTERIZATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE FROM ASCOPHYLLIUM NODOSUM (PHAEOPHYCEAE)1

Phosphoenolpyruvate carboxykinase (PEPCK) from Ascophyllum nodosum (L.) Le Jolis was partially purified and characterized to investigate its role in inorganic carbon assimilation in macroalgae. Inorganic carbon isotopic disequilibrium studies showed that the carboxylation of phosphoenolpyruvate utilized CO₂ rather than HCO₃^?as its source of inorganic carbon. This is consistent with the enzyme being a phosphoenolpyruvate carboxykinase rather than a phosphoenolpyruvate carboxylase. Pre-incubation with Mn²⁺alone activated PEPCK more effectively than when combinations of Mn²⁺, ADP and HCO₃^?were used as activators. Activation of PEPCK during catalysis was found not to occur. Although the activation of PEPCK reduced the K_m for CO₂ by a factor of 2.25, the value reported here of 1.084 mM CO₂ for the activated enzyme at pH 7.0 is at the top of the range of previously reported values for brown algal PEPCK. The specific activity of PEPCK was increased from 0.268 μmol·min^?1·mg^?1in the crude extract to 33.03 μmol·min^?1·mg^?1in the partially purified preparations. Whether PEPCK can act as an initial carboxylating enzyme is discussed. Triton X-100 at 0.57% (v/v) was found to be the optimum detergent and concentration for the extraction of enzymes from A. nodosum. When high concentrations of detergents -were used, a low (NH₄)₂SO₄ cut was required to remove the free detergent from solution, which was extracted by centrifugation. Q Sepharose was used to partially purify PEPCK and separate it from pyruvate kinase. Good protein separations were consistently obtained.     

ATP-dependent carbon transport in perfused Chara cells

Carbon transport across the plasma membrane, and carbon fixation were measured in perfused Chara internodal cells. These parameters were measured in external media of pH 5·5 and pH 8·5, where CO₂ and HCO₃^- are, respectively, the predominant carbon species in both light and dark conditions. Cells perfused with medium containing ATP could utilize both CO₂ and HCO₃^- from the external medium in the light. Photosynthetic carbon fixation activity was always higher at pH 5·5 than at pH 8·5. When cells were perfused either with medium containing hexokinase and 2-deoxyglucose to deplete ATP from the cytosol (HK medium) or with medium containing vanadate, a specific inhibitor of the plasma membrane H⁺_-ATPase (V medium), photosynthetic carbon fixation was strongly inhibited at both pH 5·5 and 8·5. Perfusion of cells with medium containing pyruvate kinase and phosphoenolpyruvate (PEP) to maximally activate the H⁺_-ATPase (PK medium), stimulated the photosynthetic carbon fixation activities. Oxygen evolution of isolated chloroplasts and the carbon fixation of cells supplied ¹⁴C intracellularly were not inhibited by perfusion media containing either hexokinase and 2-deoxyglucose or vanadate. The results indicate that Chara cells possess CO₂ and HCO₃^- transport systems energized by ATP and sensitive to vanadate in the light. In the dark, intact cells also fix carbon. By contrast, in cells perfused with medium containing ATP, no carbon fixation was detected in 1 mol m ^-3 total dissolved inorganic carbon (TDIC) at pH 8·5. By increasing TDIC to 10 mol m^-3, dark fixation became detectable, although it was still lower than that of intact cells at 1mol m^-3 TDIC. Addition of PEP or PEP and PEP carboxylase to the perfusion media significantly increased the dark-carbon fixation. Perfusion with vanadate had no effect on the dark-carbon fixation.     

PHOSPHOENOLPYRUVATE CARBOXYKINASE ACTIVITY IN ASCOPHYLLUM NODOSUM (PHAEOPHYCEAE)1

PEP-dependent⁴ CO₂-fixation by extracts of Ascophyllum nodosum (L.) Le Jol. is reported. The carboxylation of PEP is Mn²⁺ dependent and ATP is shown to be a product. IDP was found to be less efficient as a phosphate acceptor than ADP and 3-mercaptopicolinic acid inhibited the carboxylation reaction. Extracts decarboxylated OAA only in the presence of ATP and had high activities of MDH and GOT. This evidence, together with the probable absence of PEPC, PEPCTrP, and PC in A. nodosum extracts, favors the view that PEPCK is responsible for the light-independent CO₂-fixation observed in this alga.     

THE DISTRIBUTION OF PHOTOSYNTHATE IN ASCOPHYLLUM NODOSUM AS IT RELATES TO EPIPHYTIC POLYSIPHONIA LANOSA12

The brown alga Ascophyllum nodosum is not a major source of organic carbon for its epiphytic red alga Polysiphonia lanosa. Plants pulse-labeled for 24–25 hr with NaH¹⁴CO₃ were examined for exudation and translocation. The maximum amount of radioactive carbon compounds lost from A. nodosum during this experimental period was less than 0.3% of the total ¹⁴C fixed by the alga, and of this amount, only 5% could, have moved through the frond. The remaining fraction of the ¹⁴C lost from the thallus was released into the water. The total exudate from A. nodosum was collected for 1 week in a series of flasks of filtered seawater changed at 12-hr intervals corresponding with the beginning of the light and dark periods, respectively. During 7 days at 15 C only 1.5% of the total ¹⁴C originally fixed had been released as radioactive organic carbon, whereas at 5 C, 0.6% of the total ¹⁴C fixed was found in the medium. No significant difference in the rate of exudation of organic ¹⁴C was observed in light or darkness. After fractionation of the exudate it was found that only 10% of the radioactivity in these exudates was composed of organic acids or amino acids. P. lanosa, on the other hand, is perfectly capable of fixing its own carbon. The photosynthetic rates measured by H¹⁴CO₃ uptake confirm the observations of Bidwell: 3.96 mg CO₂/g/hr (0.09 m mole/g/hr).     

AMMONIUM STIMULATION OF DARK CARBON FIXATION AS AN INDICATOR OF NITROGEN DEFICIENCY IN PHYTOPLANKTON: POTENTIAL ERRORS CAUSED BY AMMONIUM-OXIDIZING BACTERIA1

Stimulation of dark fixation of carbon by NH₄⁺ is often used as an indicator of phytoplankton N deficiency. This assay is based on the influence of available NH₄⁺ on anaplerotic CO₂ fixation by algae. However, carbon fixation by chemoautotrophic NH₄⁺-oxidizing bacteria may also be stimulated by NH₄⁺ enrichment, a process that can mask the algal response in natural communities. NH₄⁺ addition enhanced dark carbon fixation up to 300%, relative to unamended controls, in organisms collected on a 0.7-μm retention filter in oligotrophic Flathead Lake, Montana, but the effect was not detectable in the presence of nitrapyrin, an inhibitor of NH₄⁺-oxidizing bacteria. Dark carbon fixation was enhanced with addition of NH₄⁺ in organisms retained on 2-μm filters (which should allow passage of most bacteria). NH₄⁺ stimulated dark carbon fixation in N-deficient axenic cultures of Chlamydomonas reinhardtii Dang but not in N-replete cultures in both the presence and absence of nitrapyrin. Application of nitrapyrin or size fractionation treatments, to separate the processes of dark carbon fixation by nitrifiers and phytoplankton, may improve the efficacy of assays using NH₄⁺ stimulation of dark carbon fixation to specifically indicate N deficiency in natural algal communities.     

Enhanced Dark CO(2) Fixation by Preilluminated Chlorella pyrenoidosa and Anacystis nidulans

The products of short time photosynthesis and of enhanced dark ¹⁴CO₂ fixation (illumination in helium prior to addition of ¹⁴CO₂ in dark) by Chlorella pyrenoidosa and Anacystis nidulans were compared. Glycerate 3-phosphate, phosphoenolpyruvate, alanine, and aspartate accounted for the bulk of the ¹⁴C assimilated during enhanced dark fixation while hexose and pentose phosphates accounted for the largest fraction of isotope assimilated during photosynthesis. During the enhanced dark fixation period, glycerate 3-phosphate is carboxyl labeled and glucose 6-phosphate is predominantly labeled in carbon atom 4 with lesser amounts in the upper half of the C₆ chain and traces in carbon atoms 5 and 6. Tracer spread throughout all the carbon atoms of photosynthetically synthesized glycerate 3-phosphate and glucose 6-phosphate. During the enhanced dark fixation period, there was a slow formation of sugar phosphates which subsequently continued at 5 times the initial rate long after the cessation of ¹⁴CO₂ uptake. To explain the kinetics of changes in the labelling patterns and in the limited formation of the sugar phosphates during enhanced dark CO₂ fixation, the suggestion is made that most of the reductant mediating these effects did not have its origin in the preillumination phase.

It is concluded that a complete photosynthetic carbon reduction cycle operates to a limited extent, if at all, in the dark period subsequent to preillumination.

     

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.     

The effect of high levels of carbon dioxide on dark respiration and growth of plants

Abstract Raising ambient levels of CO₂ during the night, between 350 and 950cm³m^?3, reduced the dark respiration rate of Medicago sativum seedlings. The percentage effect was greater for maintenance respiration than for dark respiration as a whole, and when the plants were in a low photosynthate status. Twenty-four h carbon balance studies confirmed a reduction in night time respiration and an increase of net carbon gain when night time [CO₂] was high. Growth experiments showed a small but significant increase of dry weight in Medicago sativum seedlings exposed to high [CO₂] (～ 1200 cm³m^?3) at night. This effect was greater for plants grown with Rhizobium nodules than for plants grown with nitrate in the absence of Rhizobium. A similar, but smaller and statistically non-significant effect of high night time [CO₂] on growth was found for Xanthium strumarium seedlings. The significance of these findings is discussed in relation to the rising CO₂ content of the atmosphere.     

Stimulation of nitrogenase activity and photosynthesis in some cyanobacteria by glyoxylate

Abstract The effect og glyoxylate on nitrogenase activity (C₂H₂ reduction) and photosynthesis (H¹⁴CO₃ fixation and O₂ evolution) was in vestigated in the three heterocystous cyanobacteria Anabaena cylindrica, A. variabiltis and N. muscorum. Glyoxylate had virtually no effect on the rate of dark respiration and was unable to sustain photoheterotrophic growth, though some slight stimulation (= 30%) of photorophic growth was noted. A considerable stimulation of both nitrogenase and photosynthetic activities was observed in presence of glyoxylate. In the light the stimulation increased with time up to about 15-25 h after adding optimal concentrations of 4–6 mM glyoxylate. Placing glyoxylate treated samples in the dark or adding DCMU (30 μM) in the light, showed that glyoxylate initially supported significantly higher nitrogenase activity than did samples in absence of glyoxylate. However, after a prolonged incubation in the dark or in presence of DCMU glyoxylate is unable to relieve the adverse effects of such conditions. The stimulation of the nitrogenase activity was even more pronounced when the glyoxylate was added to cells preincubated in the dark (“carbon starved”) than for cells kept constantly in light. The results suggest that glyoxylate, or a metabolite, may act as an inhibitor of cyanobacterial photorespiration and this hypothesis is discussed.     

The analysis of photosynthesis in air and water of Ascophyllum nodosum (L.) Le Jol.

Summary The photosynthetic characteristics for the intertidal macroalga Ascophyllum nodosum were examined in air and water. Under ambient conditions of temperature (10° C) inorganic carbon concentrations (15.63 mmol CO₂ m^-3 or 2.0 mol TIC m^-3) and light (500 mol photons m^-2 s^-1) photosynthesis was slightly greater by the exposed alga than by the submerged alga. In both environments photosynthesis was light saturated at 200 mol photons m^-2 s^-1. The relationship between CO₂ concentration and photosynthesis in air could be accurately analysed using Michaelis-Menten kinetics, although the range of concentrations used were not saturating. In contrast the application of the Lineweaver-Burk and Woolf plots to aquatic photosynthesis was not suitable as the experimental data was similar to the Blackman type curves and not rectangular hyperbolae. This was reflected by the applicability of the Hill-Whittingham equation to describe the photosynthesis curves. The effect of unstirred layers and other limiting factors is discussed in relation to the kinetic parameters, V _max and K _m.     

Sulphate transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

Synechococcus R-2 (PCC 1942) actively accumulates sulphate in the light and dark. Intracellular sulphate was 1.35 ± 0.23 mol m^?3 (light) and 0.894 ± 0.152 mol m^?3 (dark) under control conditions (BG-11 media: pH_o, 7.5; [SO₄^2?]_o, 0.304 mol m^?3). The sulphate transporter is different from that found in higher plants: it appears to be an ATP-driven pump transporting one SO₄^2?/ATP [ΔμSO₄^2?_i,o=+ 27.7 ± 0.24 kJ mol^?1 (light) and + 24 ± 0.34 kj mol^?1 (dark)]. The rate of metabolism of SO₄^2?at pH_o, 7.5 was 150 ± 28 pmol m^?2 s^?1 (n = 185) in the light but only 12.8 ± 3.6 pmol m^?2 s^?1 (n = 61) in the dark. Light-driven sulphate uptake is partially inhibited by DCMU and chloramphenicol. Sulphate uptake is not linked to potassium, proton, sodium or chloride transport. The alga has a constitutive over-capacity for sulphate uptake [light (n= 105): K_m= 0.3 ± 0.1 mmol m^?3, V_max, = 1.8 ± 0.6 nmol m^?2 s^?1; dark (n= 56): K_m= 1.4 ± 0.4 mmol m^?3, V_max= 41 ± 22 pmol m^?2 s^?1]. Sulphite (SO₃^2?) was a competitive inhibitor of sulphate uptake. Selenate (SeO₄^2?) was an uncompetitive inhibitor.     

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum

Nitrate-limited chemostat cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) were used to determine the effects of nitrogen addition on photosynthesis, dark respiration, and dark carbon fixation. Addition of NO₃⁻ or NH₄⁺ induced a transient suppression of photosynthetic carbon fixation (70 and 40% respectively). Intracellular ribulose bisphosphate levels decreased during suppression and recovered in parallel with photosynthesis. Photosynthetic oxygen evolution was decreased by N-pulsing under saturating light (650 microeinsteins per square meter per second). Under subsaturating light intensities (<165 microeinsteins per square meter per second) NH₄⁺ addition resulted in O₂ consumption in the light which was alleviated by the presence of the tricarboxylic acid cycle inhibitor fluoroacetate. Addition of NO₃⁻ or NH₄⁺ resulted in a large stimulation of dark respiration (67 and 129%, respectively) and dark carbon fixation (360 and 2080%, respectively). The duration of N-induced perturbations was dependent on the concentration of added N. Inhibition of glutamine 2-oxoglutarate aminotransferase by azaserine alleviated all these effects. It is proposed that suppression of photosynthetic carbon fixation in response to N pulsing was the result of a competition for metabolites between the Calvin cycle and nitrogen assimilation. Carbon skeletons required for nitrogen assimilation would be derived from tricarboxylic acid cycle intermediates. To maintain tricarboxylic acid cycle activity triose phosphates would be exported from the chloroplast. This would decrease the rate of ribulose bisphosphate regeneration and consequently decrease net photosynthetic carbon accumulation. Stoichiometric calculations indicate that the Calvin cycle is one source of triose phosphates for N assimilation; however, during transient N resupply the major demand for triose phosphates must be met by starch or sucrose breakdown. The effects of N-pulsing on O₂ evolution, dark respiration, and dark C-fixation are shown to be consistent with this model.     

High-light effects on CO2 fixation gradients across leaves

Chlorophyll fluorescence and internal patterns of ¹⁴CO₂ fixation were measured in sun and shade leaves of spinach after treatment with various light intensities. When sun leaves were irradiated with 2000μmol m^?2 s^?1 for 2h, F_V/F_M decreased by about 15%, but ¹⁴CO₂ fixation was unaffected, whereas shade leaves exhibited a 21% decrease in F_v/F_M and a 25% decrease in ¹⁴CO₂ fixation. Irradiation of sun and shade leaves with 4000μmol m^?1 for 4 h decreased F_V/F_M by 30% in sun leaves and 40% in shade leaves, while total ¹⁴CO₂ fixation decreased by 41% in sun leaves and 55% in shade leaves. After light treatment, gradients of CO₂ fixation across leaves were determined by measuring ¹⁴CO₂ fixed in paradermal leaf sections after a 10s pulse of ¹⁴CO₂. Gradients of ¹⁴CO₂ fixation in control sun and shade leaves were identified when expressed on a relative basis and normalized for leaf depth. Treatment of leaves with 2000 μmol PAR m^?2 s^?1 for 2h did not after patterns of carbon fixation across sun leaves, but slightly altered the pattern in shade leaves. In contrast, treatment of sun and shade leaves with 4000μmol m^?2 s^?1 for 4h decreased carbon fixation more in the palisade mesophyll cells than in the spongy mesophyll cells of sun and shade leaves, and fixation in medial tissue of shade leaves was dramatically decreased compared to the adaxial and abaxial tissue. The interaction between leaf anatomy and biochemical parameters involved in tolerance to photoinhibition in spinach is discussed.     

Inorganic carbon fixation and metabolism in maize roots as affected by nitrate and ammonium nutrition

The effects of NO^?₃ and NH⁺₄ nutrition on the rates of dark incorporation of inorganic carbon by roots of hydroponically grown Zea mays L. cv. 712 and on the metabolic products of this incorporation, were determined in plants supplied with NaH¹⁴CO₃ in the nutrient solution. The shoots and roots of the plants supplied with NaH¹⁴CO₃ in the root medium for 30 min were extracted with 80%; (v/v) ethanol and fractionated into soluble and insoluble fractions. The soluble fraction was further separated into the neutral, organic acid, amino acid and non-polar fractions. The amino acid fraction was then analyzed to determine quantities and the ¹⁴C content of its individual components. The rates of dark incorporation of inorganic carbon calculated from H¹⁴CO^?₃ fixation and attributable to the activity of phosphoenolpyuvate carboxylase (EC 4.1.1.31), were 5-fold higher in ammonium-fed plants than in nitrate-fed plants after a 30-min pulse of ¹⁴C. This activity forms a small, but significant component of the carbon budget of the root. The proportion of ¹⁴C located in the shoots was also significantly higher in ammonium-fed plants than in nitrate-fed plants, indicating more rapid translocation of the products of dark fixation to the shoots in plants receiving NH⁺/sp₄ nutrition. Ammonium-fed plants favoured incorporation of ¹⁴C into amino acids, while nitrate-fed plants allocated relatively more ¹⁴C into organic acids. The amino acid composition was also dependent on the type of nitrogen supplied, and asparagine was found to accumulate in ammonium-fed plants. The ¹⁴C labelling of the amino acids was consistent with the diversion of ¹⁴C-oxaloacetate derived from carboxlyation of phosphoenolpyruvate into the formation of both asparatate and glutamate. The results support the conclusion that inorganic carbon fixation in the roots of maize plants provides an important anaplerotic source of carbon for NH⁺₄ assimilation.     

Crassulacean acid metabolism (CAM) in Kalanchoë daigremontiana: Temperature response of phosphoenolpyruvate (PEP)-carboxylase in relation to allosteric effectors

Net CO₂ dark fixation of Kalanchoë daigremontiana varies with night temperature. We found an optimum of fixation at about 15° C; with increasing night temperature fixation decreased. We studied the temperature dependence of the activity of phosphoenolpyruvate (PEP)-carboxylase, the key enzyme for CO₂ dark fixation. We varied the pH, the substrate concentration (PEP), and the L-malate and glucose-6-phosphate (G-6-P) concentration in the assay. Generally, lowering the pH and reducing the amount of substrate resulted in an increase in activation by G-6-P and in an increase in malate inhibition of the enzyme. Furthermore, malate inhibition and G-6-P activation increased with increasing temperature. Activity measurements between 10° C and 45°C at a given concentration of the effectors revealed that the temperature optimum and maximum activities at that optimum varied with the effector applied. Under the influence of 5 mol m^-3 L-malate the temperature optimum and maximum activity dropped drastically, especially when the substrate level was low (at 0.5 mol m^-3 PEP from 32° C to 20° C). G-6-P raised the temperature optimum and maximum activity when the substrate level was low. If both malate and G-6-P were present, intermediate values were measured. We suggest that changes in metabolite levels in K. daigremontiana leaves can alter the temperature features of PEP-carboxylase so that the observed in vivo CO₂ dark fixation can be explained on the basis of PEP-carboxylase activity.Abbreviations PEP-c phosphoenolpyruvate carboxylase - CAM crassulacean acid metabolism - PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate     

HCO3− and CO2 leakage from Synechococcus UTEX 625

The leakage of various inorganic carbon species from air-grown cells of Synechococcus UTEX 625 was investigated after a light to dark transition or during a light period using a mass spectrometer under a wide variety of experimental conditions. Total inorganic carbon efflux and CO₂ efflux during the initial period of darkness were measured with or without carbonic anhydrase in the reaction medium respectively. The HCO₃^? efflux after a light to dark transition was estimated by difference. Carbon dioxide efflux in the light was measured by inhibiting CO₂ transport with either Na₂S or COS₃ or quenching the ¹³C inorganic carbon transport by the addition of ¹²C inorganic carbon in excess. In cells in which CO₂ fixation was inhibited, when only the HCO₃^? transport system was fully operative, CO₂ effluxed continuously during the light period at a rate equal to about 25% of that in darkness. When only the CO₂ transport system was operative, HCO₃^? effluxed during the light period. The difference between the light and dark efflux rates was consistent with a 0.6 unit decrease in the intracellular pH upon darkening the cells. The permeabilities of the cell for CO₂ (2.94 ± 0.14 ± 10^?8ms^?1; mean ± SE, n=137) and HCO₃^? (1.4–1.7 ± 10^?9 ms^?1) were calculated.     

The active uptake of carbon dioxide by the marine diatoms Phaeodactylum ticornutum and Cyclotella sp.

Mass spectromelry has been used to investigate the uptake of CO₂ by two marine diatoms, Phaeodactylum tricornutum and Cyclotella sp. The time course of CO₂ formation in the dark after addition of 100 mmol m^?3 dissolved inorganic carbon (DIC) to cell suspensions showed that external carbonic anhydrase (CA) was not present in cells of P. tricornutum but was present in Cyclotella sp. In the absence of external CA, or when it was inhibited by 5 mmol m^?3 acetazolamide, cells of both species preincubated with 100 mmol m^?3 DIG rapidly depleted almost all of the free CO₂ (3·2mmol m^?31 at pH7·5) from the suspending medium within seconds of illumination and prior to the onset of steady-state photosynthesis. Addition of bovine CA quickly restored the HCO₃^?–CO₂ equilibrium in the medium, indicating that the initial depletion of CO₂ resulted from the selective uptake of CO₂ rather than uptake of all DIG species. Transfer of cells to the dark caused a rapid increase in the CO₂ concentration in the medium, largely as a result of the efflux of unfixed inorganic carbon from the cells. The measured CO₂ uptake rates for both species accounted for 50% of the total DIG uptake at HCO₃^?–CO₂ equilibrium, indicating that HCOHCO₃^? was also being taken up. These results indicate that both Phaeodactylum tricornutum and Cyclotella sp. have the capacity to transport CO₂ actively against concentration and pH gradients.     

Pod photosynthesis and seed dark CO2 fixation support oil synthesis in developingBrassica seeds

Rate of photosynthesis and activities of photosynthetic carbon reduction cycle enzymes were determined in pods (siliqua), whereas rate of dark CO₂ fixation, oil content and activities of enzymes involved in dark CO₂ metabolism were measured in seeds ofBrassica campestris L. cv. Toria at different stages of pod/seed development. The period between 14 and 35 days after anthesis corresponded to active phase of seed development during which period, seed dry weight and oil content increased sharply. Rate of pod photosynthesis and activities of photosynthetic carbon reduction cycle enzymes were maximum in younger pods but sufficiently high levels were retained up to 40 days after anthesis. The rate of dark¹⁴CO₂ fixation in seeds increased up to 21 days after anthesis and declined thereafter but maintaining sufficiently high rates till 35 days after anthesis. Similarly various enzymes viz., phosphoenolpyruvate carboxylase, NAD⁺-malate dehydrogenase and NADP⁺-malic enzyme, involved in dark CO₂ metabolism retained sufficient activities during the above period. These enzyme activities were more than adequate to maintain the desired supply of malate which mainly arises from dark CO₂ fixation in seeds and further translocated to leucoplasts for onward synthesis of fatty acids. Enzyme localization experiments revealed phosphoenolpyruvate carboxylase and enzymes of sucrose metabolism to be present only in cytosol, whereas enzymes of glycolysis were present both in cytosolic and leucoplastic fractions. These results indicated that oil synthesis in developingBrassica seeds is supported by pod photosynthesis and dark CO₂ fixation in seeds as the former serves as the source of sucrose and the latter as a source of malate