Effect of Oxygen on Photosynthetic 14CO2 Fixation in Chroomonas sp. (Cryptophyta) II. Effects of Inhibitors, Uncouplers and an Artificial Electron Mediator on the Inhibition of 14CO2 Fixation by Anaerobiosis

In "air-grown" Chroomonas sp. cells, low concentrations of DCMU(less than 0.1 µM) could prevent the inhibition of ¹⁴CO₂fixation by anaerobiosis under light-saturating conditions (morethan 40 W.m^–2), with phenazine methosulfate showing asimilar effect. Antimycin A, carbonyl cyanide m-chlorophenylhydrazone(CCCP), and N,N'-dicyclohexylcarbodiimide strongly inhibitedanaerobic photosynthesis at concentrations which did not significantlyinhibit the rate under 2% O₂ at high light intensity (200 W.m^–2),although 0.2 µM CCCP stimulated the rate under 2% O₂ tosome extent. On the other hand, KCN inhibited the rate muchmore strongly under 2% O₂ than N₂, although it inhibited therate very strongly at concentrations above 5 µM both underN₂ and 2% O₂. These results suggest that the inhibition of photosynthetic¹⁴CO₂ fixation by anaerobiosis in this alga result from ATPdeficiency caused by over-reduction of electron carriers ofthe cyclic electron flow and that oxygen can prevent the over-reduction.Cyclic electron flow seems to be necessary to provide additionalATP for CO₂ reduction under anaerobic conditions, although itseems to be less necessary under aerobic conditions. (Received July 21, 1983; Accepted January 23, 1984)     

Red Light-Dependent CO(2) Uptake and Oxygen Evolution in Guard Cell Protoplasts of Vicia faba L.: Evidence for Photosynthetic CO(2) Fixation

Suspensions of dark-adapted guard cell protoplasts of Vicia faba L. alkalinized their medium in response to irradiation with red light. The alkalinization peaked within about 50 minutes and reached steady state shortly thereafter. Simultaneous measurements of O₂ concentrations and medium pH showed that oxygen evolved in parallel with the red light-induced alkalinization. When the protoplasts were returned to darkness, they acidified their medium and consumed oxygen. Both oxygen evolution and medium alkalinization were inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In photosynthetically competent preparations, light-dependent medium alkalinization is diagnostic for photosynthetic carbon fixation, indicating that guard cell chloroplasts have that capacity. The striking contrast between the responses of guard cell protoplasts to red light, which induces alkalinization, and that to blue light, which activates proton extrusion, suggests that proton pumping and photosynthesis in guard cells are regulated by light quality.     

14CO2 Fixation, Translocation, and Carbon Metabolism in Rapidly Expanding Leaves ofPopulus deltoides

To examine ¹⁴CO₂ fixation, potential translocation, and carbonflow among leaf chemical fractions of young developing leaves,the shoot tip of 24-leaf cottonwood (Populus deltoides Bartr.ex. Marsh) plants were cut off under water, placed in artificialxylem sap, and treated with ¹⁴CO₂ in continuous and pulse-chaseexperiments. Additional leaves on whole plants were spot treatedon the lamina tip to follow export from the tip only. The analysedleaves ranged in age from leaf plastochron index(LPI) –5to 3, the spot treated leaves from LPI 2 to 5. After 30 minfixation, the specific activity in the lamina tip increasedlinearly with leaf age from LPI –5 to 1 (0.5 to 4.5 kBqmg^–1). Specific activity in the lower lamina increasedslowly with leaf age and did not reach 500 kBq mg^–1 untilLPI –1. Total ¹⁴CO₂ fixed in the lower lamina exceededthat fixed in the tip by LPI –2 because of the large amountof tissue present in the lower lamina. Although the lamina tipfixed high levels of ¹⁴CO₂, pulse-chase studies coupled withautoradiography indicated no vein loading or translocation fromthe tip until about LPI 4 or 5. The ¹⁴C fixed in both tip andlower lamina was incorporated at the site of fixation and wasnot distributed to younger tissue or translocated from the lamina.Although the percentage distribution (¹⁴C in each chemical fractioncompared with the total in all fractions) of ¹⁴C among certainchemical fractions, e.g. sugars, amino acids and proteins, indicatedthat the mesophyll of the tip was more mature than the lowerlamina, physiologically both leaf sectors were immature basedon the expected ¹⁴C distribution in mature tissue. Informationfrom this and other studies indicates that the extreme tip ofa developing cottonwood leaf first begins to export photosynthateabout LPI 4 or 5 on a 24-leaf plant. The first photosynthatetranslocated may be incorporated into the vascular tissues andmesophyll directly below the tip. However, as the tip continuesto mature photosynthate is translocated past the immature lowerlamina into the petiole and out of the leaf. Populus deltoides Bartr. ex. Marsh, eastern cottonwood, translocation, leaf development, ¹⁴C fixation, carbon metabolism     

Photosynthetic CO(2) Fixation at Air Levels of CO(2) by Isolated Spinach Chloroplasts

A system has been developed for the study of photosynthetic CO₂ fixation by isolated spinach chloroplasts at air levels of CO₂. Rates of CO₂ fixation were typically 20 to 60 micromoles/milligrams chlorophyll per hour. The rate of fixation was linear for 10 minutes but then declined to less than 10% of the initial value by 40 minutes. Ribulose 1,5-bisphosphate (RuBP) levels remained unchanged during this period, indicating that they were not the cause for the decline. The initial activity of the RuBP carboxylase in the chloroplast was high for 8 to 10 minutes and then declined similar to the rate of CO₂ fixation, suggesting that the decline in CO₂ fixation may have been caused by deactivation of the enzyme.     

Photosynthetic Carbon Metabolism in Photoautotrophic Cell Suspension Cultures Grown at Low and High CO(2)

Photosynthetic carbon metabolism was characterized in four photoautotrophic cell suspension cultures. There was no apparent difference between two soybean (Glycine max) and one cotton (Gossypium hirsutum) cell line which required 5% CO₂ for growth, and a unique cotton cell line that grows at ambient CO₂ (660 microliters per liter). Photosynthetic characteristics in all four lines were more like C₃ mesophyll leaf cells than the cell suspension cultures previously studied. The pattern of ¹⁴C-labeling reflected the high ratio of ribulosebisphosphate carboxylase to phosphoenolpyruvate carboxylase activity and showed that CO₂ fixation occurred primarily by the C₃ pathway. Photorespiration occurred at 330 microliters per liter CO₂, 21% O₂ as indicated by the synthesis of high levels of ¹⁴C-labeled glycine and serine in a pulse-chase experiment and by oxygen inhibition of CO₂ fixation. Short-term CO₂ fixation in the presence and absence of carbonic anhydrase showed CO₂, not HCO₃⁻, to be the main source of inorganic carbon taken up by the low CO₂-requiring cotton cells. The cells did not have a CO₂-concentrating mechanism as indicated by silicone oil centrifugation experiments. Carbonic anhydrase was absent in the low CO₂-requiring cotton cells, present in the high CO₂-requiring soybean cell lines, and absent in other high CO₂ cell lines examined. Thus, the presence of carbonic anhydrase is not an essential requirement for photoautotrophy in cell suspension cultures which grow at either high or low CO₂ concentrations.     

Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: II. Metabolism of Products of CO(2) Fixation after Different Illumination Times

The metabolism of fixed ¹⁴CO₂ and the utilization of the C-4 carboxyl of malate and aspartate were examined during photosynthetic induction in Flaveria trinervia, a C₄ dicot of the NADP-malic enzyme subgroup. Pulse/chase experiments indicated that both malate and aspartate appeared to function directly in the C₄ cycle at all times during the induction period (examined after 30 seconds, 5 minutes and 20 minutes illumination). However, the rate of loss of ¹⁴C-label from the C-4 position of malate plus aspartate was relatively slow after 30 seconds of illumination, compared to treatments after 5 or 20 minutes of illumination. Similarly, the appearance of label in other photosynthetic products (e.g. 3-phosphoglycerate, sugar phosphates, alanine) during the chase periods was generally slower after only 30 seconds of leaf illumination, compared to that after 5 of 20 minutes illumination. This may be due to the lower rate of photosynthesis after 30 seconds illumination. The appearance of label in carbons 1→3 of each C₄ acid during the chase periods was relatively slow after either 30 seconds or 5 minutes illumination, while there was a relatively rapid accumulation of label in carbons 1→3 of both C₄ acids after 20 minutes illumination. Thus, while the turnover rate of the ¹⁴C-4 label in both C₄ acids increased only during the first 5 minutes of the induction period, only later during induction is there an increased rate of appearance of label in other carbon atoms of the C₄ acids. The implied source of ¹⁴C for labeling of the 1→3 positions of the C₄ acids is an apparent carbon flux from 3-phosphoglycerate of the reductive pentose phosphate pathway to phosphoenolpyruvate of the C₄ cycle.     

The Role of Cyclic and Pseudocyclic Photophosphorylation in Photosynthetic 14CO2 Fixation in Hydrodictyon africanum

Experiments are reported in which the effects on photosynthesisof various inhibitors of cyclic photophosphorylation were investigated.These inhibitors, generally had only a small inhibitory effecton photosynthesis, and the inhibition was not increased by conditionswhich inhibit pseudocyclic photophosphorylation. These inhibitorsdo not inhibit the Emerson enhancement effect. From these resultsit was concluded that photosynthesis does not need any ATP otherthan that produced in non-cyclic photophosphorylation. The effectsof these inhibitors on active K influx in light-anaerobic conditionsin the presence or absence of CO₂ suggest that some of the ATPproduced by non-cyclic photophosphorylation can be used to supportactive K influx. The results are discussed in relation to themechanism of the Emerson effect, the stoichiometry of non-cyclicphotophosphorylation, and the ATP requirements for autotrophicgrowth.     

Effect of Methionine Sulfoximine on Photosynthetic Carbon Metabolism in Wheat Leaves

Methionine sulfoximine (MSO) greatly reduced the carbon dioxideexchange rate (CER) of detached wheat (Triticum aestivvm L.cv Roland) leaves in 21% O₂, but only slightly reduced it in2% O₂. A supply of 50 mM NH₄Cl had little effect on the CERirrespective of the O₂ concentration. A simultaneous additionof glutamine and MSO protected against the inhibition of photosynthesisto a considerable extent and caused the accumulation of moreNH₃ than did the addition of MSO alone. Fixation of ¹⁴CO₂ in wheat leaves was inhibited by MSO treatmentin 22% O₂, and there was decreased incorporation of ¹⁴G intoamino acids and sugars and increased label into acid fractions.The addition of MSO and glutamine together eliminated the effectof MSO on the photosynthetic ¹⁴CO₂ fixation pattern. NH₄Cl stimulatedthe synthesis of amino acids from ¹⁴CO₂, especially the synthesisof serine in 22% O₂. Our observations show that factors other than the uncouplingof photophosphorylation by accumulated NH₃ may be responsiblefor the early stage of photosynthesis inhibition by MSO underphotorespiratory conditions. ¹Present address: Department of Agricultural Chemistry, KyushuUniversity, Fukuoka 812 Japan. ²Also at U.S. Department of Agriculture, Agricultural ResearchService, Urbana, Illionois 61801, U.S.A. (Received September 13, 1983; Accepted February 2, 1984)     

Enhanced Dark Carbon Dioxide Fixation in Maize: Effect of the Oxygen Concentration during Preillumination on CO(2) Uptake and the Intramolecular Labeling Pattern of Malate and Aspartate

The enhanced dark CO₂ uptake after a preillumination period under varying O₂ concentrations has been measured with maize, a C₄ plant. For comparison the same study has been conducted with tomato, a C₃ plant. Increasing the O₂ concentration during preillumination inhibits by 70% the subsequent dark CO₂ uptake in tomato but stimulates 2-fold this CO₂ uptake in maize. The O₂ enhancement of CO₂ uptake in maize is due to the enhancement of malate and aspartate synthesis. The percentages of radioactivity incorporated in the C-4 of malate and aspartate vary from 74 to 87% when O₂ concentration during preillumination is increased from 0 to 100%.     

CO(2) Metabolism in Corn Roots. III. Inhibition of P-enolpyruvate Carboxylase by l-malate

Phosphoenolpyruvate carboxylase was purified from corn root tips about 80-fold by centrifugation, ammonium sulfate fractionation, and anion exchange and gel filtration chromatography. The resulting preparation was essentially free from malate dehydrogenase, isocitrate dehydrogenase, malate enzyme, NADH oxidase, and pyruvate kinase activity. Kinetic analysis indicated that l-malate was a noncompetitive inhibitor of P-enolpyruvate carboxylase with respect to P-enolpyruvate (K_I = 0.8 mm). d-Malate, aspartate, and glutamate inhibited to a lesser extent; succinate, fumarate, and pyruvate did not inhibit. Oxaloacetate was also a noncompetitive inhibitor of P-enolpyruvate carboxylase with an apparent K_I of 0.4 mm. A comparison of oxaloacetate and l-malate inhibition suggested that the mechanisms of inhibition were different. These data indicated that l-malate may regulate CO₂ fixation in corn root tips by a feedback or end product type of inhibition.     

CO(2) Fixation in Opuntia Roots

Nonautotrophic CO₂ metabolism in Opuntia echinocarpa roots was studied with techniques of manometry and radiometry. The roots were grown in a one-quarter strength nutrient solution for several days; the distal 2 cm was used for physiological studies. The roots assimilated significant quantities of ¹⁴CO₂ and appeared to show a crassulacean-type acid metabolism with respect to quality and quantity. Most of the ¹⁴C activity was associated with the distal portion of the elongating root indicating correlation with metabolic activity. The ¹⁴CO₂ assimilation was comparable to a crassulacean leaf succulent, but 3 times greater than that found for stem tissue of the same Opuntia species.

The rates of O₂ and CO₂ exchange and estimated CO₂ fixation were 180, 123, and 57 μl/g per hour. A respiratory quotient of 0.66 was found.

The products of ¹⁴CO₂ fixation were similar in most respects to reported experiments with leaf succulents. Equilibration of the predominant malic acid with isocitric, succinic, and fumaric acids was not evident. The latter observation was interpreted as metabolic isolation of the fixation products rather than poor citric acid cycle activity.

A rapid turnover of the fixed ¹⁴CO₂ was measured by following decarboxlyation kinetics and by product analysis after a postincubation period. The first order rate constant for the steady state release was 4.4 × 10⁻³ min⁻¹ with a half-time of 157.5 minutes. Amino acids decayed at a more rapid rate than organic acids.

     

Effect of Triacontanol on Chlamydomonas: I. Stimulation of Growth and Photosynthetic CO(2) Assimilation

Treatment of Chlamydomonas reinhardtii cells, cultured at 5% CO₂, with 1 to 1000 micrograms triacontanol (TRIA) per liter resulted in 21 to 35% increases in cell density, 7 to 31% increases in total chlorophyll, and 20 to 100% increases in photosynthetic CO₂ assimilation. The increase in CO₂ fixation with TRIA treatment occurred before, and was independent of, increases in total chlorophyll or cell number. Chlamydomonas cells responded to a broad range of TRIA concentrations that were at least one order of magnitude above the optimum concentration established for higher plants. The necessity for larger concentrations of TRIA may be due to destabilizing effects of Ca²⁺ and K⁺ present in the Chlamydomonas growth medium. These ions caused flocculation of the colloidally dispersed TRIA in apparent competition with binding of [¹⁴C]TRIA to Chlamydomonas cells. Octacosanol inhibited the effect of TRIA on photosynthetic CO₂ assimilation. TRIA treatment did not alter the distribution of ¹⁴C-label among photosynthetic products. The effect of TRIA on photosynthetic CO₂ assimilation increased with time after treatment up to 3 days. Chlamydomonas cells that had been grown at low-CO₂ (air) did not respond to TRIA, and transfer of high-CO₂ (5%) grown cells that had responded to TRIA to a low-CO₂ atmosphere resulted in a loss of the effect of TRIA. The effect of pH on photosynthetic CO₂ assimilation indicated that CO₂ is probably the species of inorganic carbon utilized by control and TRIA-treated Chlamydomonas cells.     

Photosynthetic Reactions in the Marine Alga Codium vermilara: I. CO(2) Fixation and Hill Reaction in Isolated Chloroplasts

Chloroplasts were isolated from the marine alga Codium vermilara (Siphonales). The isolated chloroplasts were active in CO₂ fixation in the light at a rate comparable to the rates obtained by fragments of thalli. Maximal rates of CO₂ fixation by isolated chloroplasts from Codium were obtained in the presence of salt or sorbitol isoosmotic with sea water. The conditions of isolation of Codium chloroplasts are much less stringent than those required for active chloroplasts from higher plants. The isolated chloroplasts comprise a homogeneous population of the intact “class I” type, as based on microscopic observations and on their inability to reduce ferricyanide unless osmotically shocked. The intact chloroplasts are able to reduce p-benzoquinone at a high rate.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : I. Coordination of CO(2) Fixation and Sucrose Synthesis

A mechanism is proposed for a feed-forward control of photosynthetic sucrose synthesis, which allows withdrawal of carbon from the chloroplast for sucrose synthesis to be coordinated with the rate of carbon fixation. (a) Decreasing the rate of photosynthesis of spinach (Spinacia oleracea, U.S. hybrid 424) leaf discs by limiting light intensities or CO₂ concentrations leads to a 2-to 4-fold increase in fructose 2,6-bisphosphate. (b) This increase can be accounted for by lower concentrations of metabolites which inhibit the synthesis of fructose 2,6-bisphosphate, such as dihydroxyacetone phosphate and 3-phosphoglycerate. (c) Thus, as photosynthesis decreases, lower levels of dihydroxyacetone phosphate should inhibit the cytosolic fructose bisphosphatase via simultaneously lowering the concentration of the substrate fructose 1,6-bisphosphate, and raising the concentration of the inhibitor fructose 2,6-bisphosphate.     

Effect of Oxygen on the Light-enhanced Dark Carbon Dioxide Fixation in Chlorella Cells

With Chlorella ellipsoidea cells, the effect of oxygen was investigated on the products of enhanced dark ¹⁴CO₂ fixation immediately following preillumination in the absence of CO₂. When the reaction mixture was made aerobic by bubbling air (CO₂-free) throughout preillumination and the following dark ¹⁴CO₂ fixation periods, the initial fixation product was mainly 3-phosphoglyceric acid. When nitrogen gas had been used instead of air, only about one-half of the total radioactivity in the initial fixation products was in 3-phosphoglyceric acid and the rest in aspartic, phosphoenolpyruvic, and malic acids. The percentage distribution of radioactivity incorporated in these initial products rapidly decreased during the rest of the dark period. Concurrent with the decrease in the initial ¹⁴CO₂ fixation products, some increase was observed in the radioactivities of the sugar phosphates. The maximal radioactivity incorporated in sugar mono- and diphosphates accounted for only 10% of total ¹⁴C, under either the aerobic or anaerobic conditions. Under anaerobic conditions most of the ¹⁴C incorporated was eventually transferred to alanine, whereas the main end products under aerobic conditions were aspartate and glutamate. The pattern of ¹⁴CO₂ fixation products was unaffected by the atmospheric condition during the period of preillumination. The preferential flow of the fixed carbon atom to alanine or aspartate depended on the presence or absence of oxygen during the period of dark CO₂ fixation.     

Diffusional Contribution to Carbon Isotope Fractionation during Dark CO(2) Fixation in CAM Plants

A mathematical model is developed which can be used to predict in vivo carbon isotope fractionations associated with carbon fixation in plants in terms of diffusion, CO₂ hydration, and carboxylation components. This model also permits calculation of internal CO₂ concentration for comparison with results of gas-exchange experiments. The isotope fractionations associated with carbon fixation in Kalanchoë daigremontiana and Bryophyllum tubiflorum have been measured by isolation of malic acid following dark fixation and enzymic determination of the isotopic composition of carbon-4 of this material. Corrections are made for residual malic acid, fumarase activity, and respiration. Comparison of these data with calculations from the model indicates that the rate of carbon fixation is limited principally by diffusion, rather than by carboxylation. Processes subsequent to the initial carboxylation also contribute to the over-all isotopic composition of the plant.     

A Differential Effect of Race T Toxin on Dark and Photosynthetic CO(2) Fixation by Thin Leaf Slices from Susceptible Corn

The effect of purified host-specific toxin from Bipolaris (Helminthosporium) maydis, race T, on dark or light-dependent CO₂ fixation was studied with thin (1 × 8 mm) corn (Zea mays L.) leaf slices supplied H¹⁴CO₃⁻. At 5 to 30 nanograms per milliliter (5 nanomolar), toxin significantly inhibited (20 to 40%) dark CO₂ fixation in susceptible (T) corn slices after either dark or light preincubations of 10-20 minutes. The same concentrations were effective to the same degree on photosynthesis, but the effect differed in that significant inhibition occurred after 25 minutes and only with light preincubation. Light preincubation without toxin did not shorten the time required for inhibition of photosynthesis after addition of toxin. Once photosynthetic inhibition was entrained, it was not reversed by subsequent periods of darkness. The results suggest the possibility that race T toxin affects two separate metabolic sites, and the data are discussed in view of currently held concepts of toxin action in susceptible tissue.     

Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase

Kinetic properties of soybean net photosynthetic CO₂ fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO₂ concentration, and O₂ concentration. With leaves, O₂ inhibition of net photosynthetic CO₂ fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO₂ fixation at higher temperatures was caused by a reduced affinity of the leaf for CO₂ and an increased affinity of the leaf for O₂. With purified ribulose 1,5-diphosphate carboxylase, O₂ inhibition of CO₂ incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O₂ sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO₂ and a slightly increased affinity of the enzyme for O₂. The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO₂ and O₂ provides further evidence that the carboxylase regulates the O₂ response of photosynthetic CO₂ fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO₂ and O₂ fixation in vivo is proposed.