A re-examination of the problem of photochemical nitrogen reduction by blue-green algae

Summary Although it was possible in the light in the absence of carbon dioxide to obtain a ratio of nitrogen fixed to oxygen evolved in nitrogen-starved cells of A. cylindrica near to 1:1.5, that quoted by other workers, ratios varying between 1:0.9 and 1:3.0 were also obtained. The amount of oxygen evolved under the same conditions by normal cells in the presence of pyruvate was increased considerably. Since the addition of pyruvate also resulted in increased carbon dioxide output in the dark with the same algal material, oxygen output in the light was attributed to the production of factors necessary for carbon assimilation.Addition of pyruvate to nitrogen-starved and normal cells in the light resulted in similar rates of oxygen evolution after an initially higher rate in the starved cells. The ratio of overall nitrogen fixed to oxygen evolved, was 1:6.6 for the starved cells and 1:6.4 for the normal cells, showing that the presence of an added substrate increased oxygen output relative to nitrogen uptake. ¹⁴CO₂ was recovered from sodium pyruvate-1-¹⁴C in flasks incubated in the dark, showing that, at least in the dark, pyruvate was decarboxylated.The interpretation of these results is that endogenous and exogenous substrates available to cells of A. cylindrica become decarboxylated and that, in the light, carbon dioxide produced may be assimilated photochemically with accompanying oxygen evolution. This interpretation has been discussed in relation to reports of photochemical nitrogen reduction in blue-green algae.     

COMPARATIVE STUDIES OF NITROGEN FIXATION AND PHOTOSYNTHESIS IN ANABAENA CYLINDRICA

Manometric methods were developed to allow short-time measurements on relatively small amounts of cell material. The methods provided measurement of the nitrogen uptake and oxygen evolution attributable to nitrogen fixation either in the presence or absence of carbon dioxide. The methods were applied to observe effects of cell history, light intensity, temperature, pH, and ammonia on nitrogen fixation and photosynthesis with 2 new and significant findings. Nitrogen fixation is markedly accelerated by a preceding period of cellular nitrogen depletion. Nitrogen fixation is depressed by light intensities greater than those required to saturate photosynthesis.     

Glycolate Stimulation of Oxygen Evolution During Photosynthesis

Glycolate and glyoxylate stimulated 100% to 300% the rate of oxygen evolution by Scenedesmus in the light in the absence of added carbon dioxide. This stimulation occurred either aerobically or anaerobically, and was sensitive to CMU. Aerobic dark respiration was stimulated 25% to 100% by glycolate. This phenomenon was best demonstrated with synchronized Scenedesmus at the stage of cell division. For glycolate stimulation of oxygen evolution, a dark preincubation of 1 minute or less was necessary. In comparative test with other compounds of metabolism and photosynthesis, the stimulation of oxygen evolution was greatest by glycolate and glyoxylate. In a proposed scheme glyoxylate serves as a terminal hydrogen acceptor from NADPH produced by photosynthesis, and it thereby stimulates oxygen evolution when carbon dioxide is not available. Transformation of glycolate to glyoxylate in these cells would have to occur in the absence of oxygen.     

Control of photosynthesis during nitrogen depletion and recovery in a non-nitrogen-fixing cyanobacterium

When cells of Synechocystis strain PCC 6308 are starved for nitrogen, the amount of stored carbohydrate increases, the phycocyanin to chlorophyll a ratio decreases, and the rates of oxygen evolution and of carbon dioxide fixation decrease. When nitrate-nitrogen is replenished, the amount of carbohydrate decreases, the rate of oxygen evolution increases immediately, preceeding the increase in phycocyanin or carbon dioxide fixation. The rate of respiration first increases and then decreases upon nitrogen addition. Nitrogen-starved cells show no variable fluorescence; variable fluorescence recovered in parallel with oxygen evolution. This suggests that photosystem II is inactive in nitrogen depleted cells and not blocked by a build up of metabolic endproducts. Since carbon dioxide fixation does not increase until two to four hours after nitrate is replenished to nitrogen starved cells, it is suggested that reducing power may first be needed within the cell for some other process than photosynthesis, such as nitrate reduction.     

Oxygen and carbon dioxide fluxes from barley shoots depend on nitrate assimilation

A custom oxygen analyzer in conjunction with an infrared carbon dioxide analyzer and humidity sensors permitted simultaneous measurements of oxygen, carbon dioxide, and water vapor fluxes from the shoots of intact barley plants (Hordeum vulgare L. cv Steptoe). The oxygen analyzer is based on a calciazirconium sensor and can resolve concentration differences to within 2 microliters per liter against the normal background of 210,000 microliters per liter. In wild-type plants receiving ammonium as their sole nitrogen source or in nitrate reductase-deficient mutants, photosynthetic and respiratory fluxes of oxygen equaled those of carbon dioxide. By contrast, wild-type plants exposed to nitrate had unequal oxygen and carbon dioxide fluxes: oxygen evolution at high light exceeded carbon dioxide consumption by 26% and carbon dioxide evolution in the dark exceeded oxygen consumption by 25%. These results indicate that a substantial portion of photosynthetic electron transport or respiration generates reductant for nitrate assimilation rather than for carbon fixation or mitochondrial electron transport.     

Studies on the Biochemistry and Fine Structure of Silica Shell Formation in Diatoms. Photosynthesis and Respiration in Silicon-Starvation Synchrony of Navicula pelliculosa

Rates of photosynthesis, measured by oxygen electrode or by ¹⁴CO₂ fixation, dark respiration and ³²P-phosphate incorporation are reported for the silicon-starvation synchrony of the fresh water diatom Navicula pelliculosa. During late exponential growth the rates were consistent with increase in carbon mass. During silicon starvation, rates of carbon dioxide fixation, oxygen evolution and ³²P incorporation fell, and the saturating light intensity decreased from 27,000 lux to 5000 lux. Reintroduction of silicon led to immediate transients in all parameters studied, followed by a prolonged increase in rate of dark respiration and a gradual increase in apparent photosynthesis. During release of daughter cells, the rates of dark respiration decreased as photosynthesis and ³²P incorporation increased. These results are discussed in relation to effects of silicon on the energy metabolism of the diatom.     

‘Respiratory protection’ of the nitrogenase in dinitrogen-fixing cyanobacteria

Several filamentous and unicellular cyanobacteria were grown photoautotrophically with nitrate or dinitrogen as N-sources, and some respiratory properties of the cells or isolated plasma (CM) and thylakoid (ICM) membranes were compared. Specific cytochrome c oxidase activities in membranes from dinitrogen-fixing cells were between 10- and 50-times higher than those in membranes from nitrate-grown cells, ICM of heterocysts but CM of unicells being mainly responsible for the stimulation. Whole cell respiration (oxygen uptake) of diazotrophic unicells paralleled increased cytochrome oxidase activities of the isolated membranes. Mass spectrometric measurements of the uptake of isotopically labeled oxygen revealed that (low) light inhibited respiration of diazotrophic unicells to a much lesser degree than that of nitrate-grown cells which indicates the prevailing (respiratory) role of CM in the former. Normalized growth yields of diazotrophic unicells grown in continuous light were significantly higher than those of cells grown in a 12/12 hrs light/dark cycle. Mass spectrometry showed that overall nitrogen uptake by the former was higher than by the latter; in particular, and in marked contrast to the time course of nitrogenase activity (acetylene reduction) there was no appreciable nitrogen uptake or protein synthesis during dark periods; likewise, there was no 14-CO₂ fixation, nor chloropholl synthesis, nor cell division in the dark. By contrast, growth in continuous light gave sustained rates of nitrogen and carbon dioxide incorporation over the whole time range. Our results will be discussed in terms of respiratory protection as an essential strategy of keeping apart nitrogenase and oxygen, either atmospheric or photosynthetically produced within the same cell.     

The Relationships between Nitrogen Fixation and Several Kinds of Energy Metabolism by Gloeocapsa sp

Gloeocapsa sp., a species of anicellular blue-green alga, fixes dinitrogen mostly under light. The energy (ATP and reductant) needed for nitrogen fixation may be provided by photoreaction and aerobic catabolism. The nitrogenase activity (acetylene reduction) in vivo was decreased under the conditions of dark and inhibition of photo-phosphorylation or oxidative phosphorylation in the light. When photosystem Ⅱ was inhibited by the presence of DCMU, nitrogenase activities in both reactions of acetylene reduction and hydrogen evolution may be muchenhanced probably due to eliminating of the damage caused by the oxygen produced in the photolysis of water. The effects of the oxygen present in the atmosphere of the reaction systemand produced by the cells are different. It is shown that some trace oxygen seems to be required for nitrogen fixation by the energy supply of aerobic actabolism and oxidative phosphorylation. While the fixation of dinitrogen was inhibited by CO or no any reducible substrate was present, 70-100% of the energy accepted by nitrogenase was evolved as hydrogen. The algal cells also showed hydrogen uptake reaction, but no enhancement of nitrogen fixation by the hydrogen uptake was found.     

Nitrite Uptake into Intact Pea Chloroplasts : II. Influence of Electron Transport Regulators, Uncouplers, ATPase and Anion Uptake Inhibitors and Protein Binding Reagents

The relationship between net nitrite uptake and its reduction in intact pea chloroplasts was investigated employing electron transport regulators, uncouplers, and photophosphorylation inhibitors. Observations confirmed the dependence of nitrite uptake on stromal pH and nitrite reduction but also suggested a partial dependance upon PSI phosphorylation. It was also suggested that ammonia stimulates nitrogen assimilation in the dark by association with stromal protons. Inhibition of nitrite uptake by N-ethylmaleimide and dinitrofluorobenzene could not be completely attributed to their inhibition of carbon dioxide fixation. Other protein binding reagents which inhibited photosynthesis showed no effect on nitrite uptake, except for p-chlormercuribenzoate which stimulated nitrite uptake. The results with N-ethylmaleimide and dinitrofluorobenzene tended to support the proposed presence of a protein permeation channel for nitrite uptake in addition to HNO₂ penetration. On the basis of a lack of effect by known anion uptake inhibitors, it was concluded that the nitrite uptake mechanism was distinct from that of phosphate and chloride/sulfate transport.     

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.     

The effect of nutrient deficiencies on phosynthesis and respiration in spinach

Summary Spinach plants were grown in nutrient-culture solutions containing reduced levels of all the macro- and micro-nutrient elements except cobalt and chlorine. The rates of photosynthesis (carbon dioxide fixation in the light expressed on a per unit chlorophyll or per unit fresh-weight basis) and respiration (carbon dioxide evolution in the dark expressed on a per unit nitrogen or per unit fresh-weight basis) for whole plants were measured using infra-red gas analysis techniques. Measurements were made when the plants displayed clear symptoms of deficiency relative to control plants. All nutrient deficiencies except iron and molybdenum depressed photosynthesis when chlorophyll was the basis of calculation; manganese-, copper-, phosphorus- and potassium-deficient plants showed the greatest depression. Alternatively when photosynthesis was calculated on a fresh weight basis calcium was the only deficiency which had no affect. Similarly when respiration was calculated on a nitrogen basis all deficiencies except iron, molybdenum and nitrogen result in depressed rates but when respiration was expressed on a fresh-weight basis potassium deficiency resulted in enhanced respiration rates and nitrogen, phosphorus, sulphur, manganese, zinc and molybdenum deficiencies resulted in reduced respiration rates.     

Effect of lectins on auxin-induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

A marine unicellular aerobic nitrogen-fixing cyanobacterium Synechococcus sp. strain Miarni BG 043511 was pretreated with different light and dark regimes in order to induce higher growth synchrony. A pretreatment of two dark and light cycles of 16 h each yielded good synchrony for 3 cell division cycles. Longer dark treatments decreased the degree of synchrony and shorter dark treatments caused irregular cell division. Once synchronous culture was established, distinct phases of cellular carbohydrate accumulation and cellular carbohydrate degradation were observed even under continuous illumination. Changes in carbohydrate content were repeated in a cyclic manner with approximately 20 h intervals, the same as the cell division cycle. This change in carbohydrate metabolism provided a good index of growth synchrony under nitrogen-fixing conditions.
Photosynthetic oxygen evolution and nitrogen fixation capabilities and their activities in near, in situ, culture conditions were measured in well synchronized cultures of this strain under continuous illumination. Distinct oscillations of both photosynthetic oxygen evolution and nitrogen fixation capabilities with ca 20-h intervals, similar to the interval of the cell division cycle, were observed for three cycles. However, the activities of photosynthetic oxygen evolution were inversely correlated with those of nitrogen fixation. During the nitrogen fixation period, net oxygen consumption was observed even in the light under conditions approximating in situ culture conditions. The phase of temporal appearance of nitrogenase activity during the cell division cycle coincided with the phase of carbohydrate net degradation. These data indicate that this unicellular cyanobacterium can grow diazotrophically under conditions of continuous illumination by the segregation of photosynthesis and nitrogen fixation within a cell division cycle.     

Evidence for the occurrence of photorespiration in synurophyte algae

The fluxes of CO(2) and oxygen during photosynthesis by cell suspensions of Tessellaria volvocina and Mallomonas papillosa were monitored mass spectrometrically. There was no rapid uptake of CO(2,) only a slow drawdown to compensation concentrations of 26 μM for T. volvocina and 18 μM for M. papillosa, when O(2) evolution ceased, indicating a lack of active bicarbonate uptake by the cells. Darkening of the cells after a period of photosynthesis did not cause rapid release of CO(2), indicating the absence of an intracellular inorganic carbon pool. However, upon darkening a brief burst of CO(2) was observed similar to the post-illumination burst characteristic of C(3) higher plants. Treatment of the cells of both species with the membrane-permeable carbonic anhydrase inhibitor ethoxyzolamide had no adverse effect on photosynthetic rate, but stimulated the dark CO(2) burst indicating the dark oxidation of a compound formed in the light. In the absence of any active accumulation of inorganic carbon photosynthesis in these species should be inhibited by O(2). This was investigated in four synurophyte species T. volvocina, M. papillosa, Synura petersenii, and Synura uvella: photosynthetic O(2) evolution rates in all four algae, measured by O(2) electrode, were significantly higher (40-50%) in media at low O(2) (4%) than in air-equilibrated (21% O(2)) media, indicating an O(2) inhibition of photosynthesis (Warburg effect) and thus the occurrence of photorespiration in these species.     

Cyclic variations of photosynthetic activity under nitrogen fixing conditions in Synchococcus RF-1

When nitrogen fixing cell cultures of Synechococcus RF-1 were subjected to an alternating lightdark regime (12 h:12 h), a cyclic decrease in the photosynthetic oxygen evolution potential was observed during the dark periods. This rhythm of net photosynthesis rate was maintained for at least two days after transition to continuous light. The decrease in net photosynthesis was accompanied by a stimulation of dark respiration. However, the magnitude of oxygen uptake was considerably smaller than the observed decrease in oxygen evolution. The photosynthetic activity of cells taken from the dark period was characterized by (i) a significantly lower quantum yield and (ii) a strong reduction in the light-saturated rate of photosynthesis. Growing the cultures on nitrate or under continuous light completely suppressed this rhythm. Protein synthesis was not necessary for the recovery of the light-saturated rate of photosynthesis during the light period. The cellular content of chlorophyll a and of phycobiliproteins did not vary between light and dark period, indicating that quantitative changes in the composition of the photosynthetic apparatus are not the basis for the observed oscillations. Regulatory modifications of the photosynthetic efficiency are proposed as an adaptation mechanism to adjust the intracellular oxygen concentration to the needs for nitrogenase activity.Abbreviation Chl chlorophyll     

Hydrogenase in pea root nodule bacteriods

Summary Hydrogen uptake has been shown to occur with pea root nodule breis and this uptake has been shown to be confined to the bacteriods. The uptake of hydrogen by washed bacteriods, in the absence of any added substrates, has been shown to be accompanied by oxygen uptake and the ratio of hydrogen uptake to oxygen uptake in these preparations has been found to be 2:1. Substrates, provided to washed bacteriods, inhibit the uptake of hydrogen and it has been found that the utilisation of substrates, as measured by carbon dioxide evolution, is inhibited by hydrogen. It is suggested that hydrogen and substrates compete for electron carriers and that electrons from the hydrogen reduce components of the electron transport pathway and ATP is produced.The action of hydrogen on nitrogen fixation in nodule breis and washed bacterioid preparations was examined and the evidence shows that some non-competitive inhibition of nitrogen fixation, by hydrogen, occurs.     

Respiration and nitrogen fixation in nodulated roots of soya bean and pea

Summary Oxygen uptake, carbon dioxide evolution and nitrogenase activity, measured either as hydrogen evolution (under argon 80%, oxygen 20%) or as the reduction of acetylene to ethylene, were assayed over the same time period by a direct mass-spectrometric method. When carbon dioxide evolution was used to estimate carbohydrate consumption, the results agreed with other work on whole plants. The RQ values obtained in these experiments were always less than 1.0 and thus the carbohydrate consumption calculated from oxygen uptake suggests that previous estimates, using carbon dioxide evolution as a measure of the cost of nitrogen fixation may be underestimates. Lag periods observed in the reduction of acetylene to ethylene suggest that there is a resistance to diffusion of gases in the root nodules.     

蓝藻Anabaena 7120光漂白细胞固氮的研究

蓝藻Anabaena 7120经光漂白后固氮活性明显下降,转入正常光照下又恢复活性。此种经光漂白的蓝藻细胞,其固氮活性对氧敏感度小,受分子氢的促进大些,而忍受CO_2和N_2抑制的浓度相对高些。其固氮活性为弱光和光合抑制剂减弱,而加入外源的碳水化合物则能提高它的固氮活性。当碳水化合物和光合抑制剂一起加入反应系统时,蓝藻光漂白细胞的固氮活性并不能受到促进。     

Light control of the respiration of exogenous glycerol in the red macroalga Grateloupia doryphora

Heterotrophic activity in macroalgae has been little studied, but the red macroalga Grateloupia doryphora is known to grow in light at a higher rate in a glycerol-containing medium than in seawater. The effects of 0·1 M exogenous glycerol in seawater (SW90-gly) on the respiration rate of G. doryphora and the role played by light were investigated. The algae pretreated for 2 h in the light and in SW90-gly evolved oxygen and fixed carbon dioxide (H¹⁴CO₃ ^?), but also evolved radioactive ¹⁴CO₂ from [¹⁴C]glycerol. The rate of oxygen evolution was lower than that of samples in seawater, due to a high respiration rate and/or a partial inhibition of photosynthesis induced by glycerol. In contrast, the rate of inorganic carbon fixation was higher in SW90-gly than in control samples in seawater, suggesting that non-photosynthetic patterns were operating. In darkness, after pretreatment in the light in SW90-gly, samples showed a high oxygen uptake rate just after the light was turned off. Twenty minutes of darkness were enough to decrease this high respiration rate to that of samples in seawater. The oxygen uptake observed in all experiments with glycerol was mitochondrial as it was inhibited by potassium cyanide and salicylhydroxamic acid (SHAM). Pretreatment of samples in the light in SW90-gly with the photosynthetic inhibitor DCMU did not inhibit ensuing dark respiration, thus providing evidence for a non-photosynthetic effect of the light. The highest dark respiration rate was observed after the samples were pretreated in monochromatic blue light in glycerol-containing media.     

Photosystem II Independent Carbon Dioxide Fixation in Plectonema boryanum During Photoautotrophic Growth Under Nitrogen Fixation Conditions

The non-heterocystous filamentous cyanobacterium Plectonema boryanum UTEX 594 grew rapidly microaerobically under nitrogen-starvation conditions in continuous high light intensity by conducting oxygenic photosynthesis and oxygen sensitive nitrogen-fixation in alternating cycles. During diazotrophic phase, the light harvesting pigment phycocyanin declined with a concomitant depression in light dependent oxygen evolution by the cyanobacterium. A substantial component of light dependent carbon dioxide fixation during diazotrophic phase was not inhibited by DCMU in spite of complete cessation of photosynthetic oxygen evolution. Endogenous-reductant dependent electron transfer to photosystem I during diazotrophic phase is postulated even during photoautotrophic growth.     

Resolution of Conflicting Signals at the Single-Cell Level in the Regulation of Cyanobacterial Photosynthesis and Nitrogen Fixation

Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N₂) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N₂ fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N₂ fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N₂ fixation and photosynthesis commonly observed. However, N₂ fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell.