Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

Can genotypes of soybean (Glycine max) selected for nitrate tolerance provide good "models" for studying the mechanism of nitrate inhibition of nitrogenase activity?

In soybeans ( Glycine max L. Merr.), high levels of soil nitrate inhibit N₂ fixation, and nitrate-tolerant symbioses have been identified within a chemically mutagenized line of cv. Bragg denoted nts382 and within the line K466, a genotype representative of a number of Korean soybean cultivars. The genotypes nts382 and K466 were examined to see if they could be used as a model system for studying the mechanism responsible for the short-term (i.e. 3-day) inhibition of specific nitrogenase activity, especially the mechanism behind the greater O₂ limitation of nodule metabolism that is characteristic of nitrate inhibition of N₂ fixation in soybean. In nts382, total nitrogenase activity (TNA = H₂ production in Ar:O₂) was inhibited to a lesser degree (48% of control) relative to Bragg (30% of control), and the nitrate-treated symbioses showed less of an O₂ limitation of nodule metabolism in nts382 than in Bragg. However, the relative proportion of O₂ limitation to the total nitrate inhibition was similar (40 and 41%) in nts382 and Bragg, respectively. Therefore, the nts382 symbioses may be useful in elucidating the general mechanism for down-regulation of nitrogenase activity in soybean, but would not be a useful model system for studying the control of O₂-limited metabolism following nitrate exposure. The effects of nitrate on TNA and on the degree of O₂ limitation of nodule metabolism were the same in K466 and a reference cultivar Maple Arrow. Consequently, the tolerance of K466 to nitrate reported previously was attributed to the ability of this symbiosis to maintain nodule biomass in the presence of nitrate, not to any ability to maintain specific nitrogenase activity in the presence of nitrate.     

Can genotypes of soybean (Glycine max) selected for nitrate tolerance provide good “models” for studying the mechanism of nitrate inhibition of nitrogenase activity?

In soybeans ( Glycine max L. Merr.), high levels of soil nitrate inhibit N₂ fixation, and nitrate-tolerant symbioses have been identified within a chemically mutagenized line of cv. Bragg denoted nts382 and within the line K466, a genotype representative of a number of Korean soybean cultivars. The genotypes nts382 and K466 were examined to see if they could be used as a model system for studying the mechanism responsible for the short-term (i.e. 3-day) inhibition of specific nitrogenase activity, especially the mechanism behind the greater O₂ limitation of nodule metabolism that is characteristic of nitrate inhibition of N₂ fixation in soybean. In nts382, total nitrogenase activity (TNA = H₂ production in Ar:O₂) was inhibited to a lesser degree (48% of control) relative to Bragg (30% of control), and the nitrate-treated symbioses showed less of an O₂ limitation of nodule metabolism in nts382 than in Bragg. However, the relative proportion of O₂ limitation to the total nitrate inhibition was similar (40 and 41%) in nts382 and Bragg, respectively. Therefore, the nts382 symbioses may be useful in elucidating the general mechanism for down-regulation of nitrogenase activity in soybean, but would not be a useful model system for studying the control of O₂-limited metabolism following nitrate exposure. The effects of nitrate on TNA and on the degree of O₂ limitation of nodule metabolism were the same in K466 and a reference cultivar Maple Arrow. Consequently, the tolerance of K466 to nitrate reported previously was attributed to the ability of this symbiosis to maintain nodule biomass in the presence of nitrate, not to any ability to maintain specific nitrogenase activity in the presence of nitrate.     

Nitrogen fixation by the unicellular cyanobacterium Gloeothece. Nitrogenase synthesis is only transiently repressed by oxygen

Abstract The extent of recovery of nitrogenase activity of Gloeothece transferred from an atmosphere of O₂ to air depended on the duration of exposure to O₂. Activity recovered at increasing rates after up to 24 h exposure to O₂ and a lag before detection of activity, present after short (1 h) exposure times, disappeared with longer exposures. Synthesis of nitrogenase de novo was implicated, since chloramphenicol, tetracycline, or repressive levels of NH⁺₄, prevented recovery of activity. Specific radioimmunoassay of the rate of synthesis of the MoFe protein of nitrogenase under O₂ correlated well with the activity measurements, and indicate that a shift from air to O₂ only transiently represses nitrogenase synthesis.     

Effects of age, supra-ambient oxygen and repeated assays on acetylene reduction and root respiration in pea

An open flow-through gas system was used to investigate the effect of plant age on nitrogenase activity in relation to root respiration (measured as CO₂ release) and supra-ambient O₂ levels in 24- to 51-day-old, nodulated Pisum sativum L. cv. Bodil. The effect of assaying plants repeatedly was also studied. The respiratory efficiency of nitrogenase [mol CO₂ (mol C₂H₄)⁻¹] and the relative decline in nitrogenase (EC 1.7.99.2) activity in response to introduction of C₂H₂ in the gas stream were unaffected by plant age. In contrast, the nitrogenase-linked respiration as a proportion of total root respiration increased with time. Accordingly, the specific respiration linked-to growth and maintenace of the noduled root system decreased with time. C₂H₂ reduction and root respiration were increased by supra-ambient O₂ levels, but the tolerance to high O₂ concentrations seemed to decrease with plant age. Repeated C₂H₂ assays on the same plants decreased their rate of growth and N accumulation: in addition, nitrogenase activity and root respiration were somewhat negatively affected. The results indicate that results from experiments with plants of different ages cannot always be directly compared, and that repeated C₂H₂ assays on the same plants should be applied with caution in physiological work.     

Effects of inorganic nitrogen compounds on the activity and synthesis of nitrogenase in Gloeothece (Nägeli) sp. ATCC 27152

Addition of 2 mM nitrite or ammonium to aerobically incubated cultures of Gloeothece rapidly inhibited N₂ fixation (measured as acetylene reduction). In contrast, 2 mM nitrate inhibited N₂ fixation less rapidly and less extensively, and often temporarily stimulated nitrogenase activity. The inhibitory effects of both nitrate and ammonium could be prevented by addition of 3 mM L-methionine-DL-sulphoximine, suggesting that the true inhibitor of N₂ fixation was an assimilatory product of ammonium rather than either ammonium or nitrate itself. The inhibition of N₂ fixation by nitrite could not, however, be prevented by addition of L-methionine-DL- sulphoximine. On the other hand, nitrite (unlike nitrate and ammonium) did not inhibit N₂ fixation in cultures incubated under a gas phase lacking oxygen. These findings suggest that the mechanism whereby nitrite inhibits N₂ fixation in Gloeothece differs from that of either nitrate or ammonium. The inhibitory effect of nitrite on N₂ fixation did not involve reduction of nitrite to nitric oxide, though nitric oxide was a potent inhibitor of nitrogenase activity in Gloeothece . Nitrate and nitrite inhibited the synthesis of nitrogenase in Gloeothece , while ammonium not only inhibited nitrogenase synthesis but also stimulated degradation of the enzyme. In addition, all three compounds favoured the appearance of the Fe-protein of nitrogenase in its larger, presumed inactive, form.     

Regulation of nitrogen fixation by nitrite and glutamine in Derxia gummosa

Abstract Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C₂H₂ reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C₂H₂ reduction had a K _i value of 16 μM. Both ammonia and glutamine inhibited N₂ fixation (C₂H₂ reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.     

Aerobic nitrogen fixation is confined to a subset of cells in the non-heterocystous cyanobacterium Symploca PCC 8002

Symploca PCC 8002 Kützing is a filamentous cyanobacterium that lacks the specialized cells, known as heterocysts, that protect nitrogenase from O₂ in most aerobic N₂-fixing cyanobacteria. Nevertheless, Symploca is able to carry out N₂ fixation in the light under aerobic conditions. When cultures were grown under light/dark cycles, nitrogenase activity commenced and increased in the light phase and declined towards zero in the dark. Immunolocalization of dinitrogenase reductase in sectioned Symploca trichomes showed that the enzyme was present only in 9% of the cells. These cells lacked any obvious mechanical protection against atmospheric O₂ and their ultrastructural characteristics were similar to those of cells that did not contain any dinitrogenase reductase. The nitrogenase-containing cells possessed carboxysomes that were rich in ribulose-1,5-bisphosphate carboxylase/oxygenase and phycoerythrin, a light harvesting pigment of PS II. This indicates that these cells had a capacity for both N₂ fixation and photosynthesis. The significance of the localization pattern for dinitrogenase reductase is discussed in the context of N₂ fixation in Symploca PCC 8002.     

Regulation of nitrate reductase in detached oat leaves by light and oxygen

Regulation of nitrate reductase (NR, EC 1.6.6.1) by oxygen concentration and light was studied in segments of oat ( Avena sativa L. cv. Suregrain) leaves, using the in vivo nitrate reductase assay. The activity of NR decreased after excision in either light or darkness; the addition of cycloheximide prevented this decrease. Treatments that increased tissue permeability (anoxia, Triton X-100) also increased NR activity. There was in general less NR activity in the light than in the dark and also less under aerobic (21–100% O₂) than under anaerobic (0.3% O₂) conditions. Treatments with antioxidants improved the activity in the light, but only at high O₂ levels (21–100% O₂).
The results suggest that NR may be regulated by inhibitory proteins synthesized in either light or darkness, by permeability changes and by light-induced oxidations that occur when O₂ is present. Oxygen may control the activity by stimulating the synthesis of inhibitory proteins in the light and in the dark and by promoting oxidation of SH-groups in the light.     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.     

Superoxide dismutase, catalase and nitrogenase activities of symbiotic Frankia (Alnus incana) in response to different oxygen tensions

Presence and activity of the enzymes superoxide dismutase (SOD) and catalase were studied in Frankia in symbiosis with Alnus incana (L.) Moench. Analysis on native PAGE gels indicated that symbiotic Frankia contained an FeSOD and catalase. The activity of the enzymes was in the same range as reported for cultured Frankia . Attempts to characterize SOD by western blots with antisera from Escherichia coli and Azotobacter vinelandii did not give clear-cut results with the antibodies used. Alnus incana plants were grown with the root system in 5, 10, 21 or 40% O₂ for up to 6 days. Nitrogenase activity, measured as ARA (acetylene reducing activity) dropped within 3 h when roots were exposed to low or high oxygen. At 40% O₂ ARA was almost completely lost while at 5 and 10% O₂ ARA decreased to 69 and 74% of the inital value, respectively, Nitrogenase activity recovered at ail oxygen tensions. Recovery rates resembled the continuous increase in ARA in plants continuosly kept at 21% O₂, and suggests that new vesicles with envelopes of appropriate thickness were formed. The ARA measurements confirm results from an earlier study where nitrogenase activity was measured as H₂ evolution. There was a tendency for increased SOD and catalase activities in Frankia from root systems exposed to 40% O₂ for 24 h but not earlier or later than this. When data from all experimental times were pooled. SOD activity increased significantly with increased oxygen tension whereas catalase activity decreased. Although ARA per plant varied with oxygen tension, there was no statistically significant correlation between ARA and SOD or between ARA and catalase. It seems that being linked to nitrogenase activity is only one role of SOD and catalase in this symbiotic Frankia .     

Role of oxygen limitation and nitrate metabolism in the nitrate inhibition of nitrogen fixation by pea

The impact of nitrate (5–15 m M , 2 to 7 days) on nitrogenase activity and nodule-oxygen limitation was investigated in nodulated, 21-day-old plants of a near-isogenic nitrate reductase-deficient pea mutant (A3171) and its wild-type parent ( Pisum sativum L. cv. Juneau). Within 2 days, 10 or 15 m M nitrate, but not 5 m M nitrate, inhibited the apparent nitrogenase activity (measured as in situ hydrogen evolution from nodules of intact plants) of wild-type plants; none of these nitrate levels inhibited the apparent nitrogenase activity of A3171 plants. Nodule-oxygen limitation, measured as the ratio of total nitrogenase activity to potential nitrogenase activity, was increased in both wild-type and A3171 plants by all nitrate treatments. By 3 to 4 days the apparent nitrogenase activity of A3171 and wild-type plants supplied with 5 m M nitrate declined to 53 to 69% of control plants not receiving nitrate. By 6 to 7 days the apparent nitrogenase activity of A3171 plants was similar to the control value whereas that of the wild-type plants continued to decline. From 3 to 7 days, no significant differences in nodule-oxygen limitation were observed between the nitrate (5 m M ) and control treatments. The results are interpreted as evidence for separate mechanisms in the initial (O₂ limitation) and longer-term (nitrate metabolism) effects of nitrate on nitrogen fixation by effectively nodulated pea.     

Hydrogen Peroxide Production by Rat Brain In Vivo

Abstract: H₂ O₂ production by rat brain in vivo was observed with a method based on the measurement of brain catalase. The administration to the rat of 3-amino-1, 2, 4-triazole, an H₂ O_2- dependent inhibitor of catalase, caused progressive inhibition of brain catalase activity in both the supernatant and pellet fractions of homogenates of the striatum and prefrontal cortex. The prevention of catalase inhibition by prior administration of ethanol confirmed that catalase inhibition in vivo was dependent upon H₂ O₂. A significant portion of the catalase (30-33%) appeared in the supernatant fraction from a slow-speed homogenization procedure and was not significantly contaminated by either erythrocytes or capillaries. In the whole homogenate, less than 6% of the catalase activity was attributed to erythrocytes. Modification of intracellular monoamine oxidase activity by either pargyline or reserpine did not change the rate of inhibition of catalase by aminotriazole. A probable interpretation of these data is that H₂ O₂ generated by mitochondrial monoamine oxidase does not reach the catalase compartment; the catalase is contained in particles described by other investigators as the microperoxisomes of brain. In studies in vitro , the production of H₂ O₂ by rat brain mitochondria with either dopamine or serotonin as substrate was confirmed.     

Characterization of extracellular oxygen consumption by the green alga Selenastrum minutum

Cells of the green alga Selenastrum minutum display a high capacity for extra-mitochondrial O₂ consumption in the presence of effectors such as salicylhydroxamic acid and/or NADH. We provide evidence that this O₂ consumption is mediated by extracellular peroxidase. Peroxidase capacity, measured as the potential for stimulation of O₂ consumption by a combination of salicylhydroxamic acid and NADH, changed over a 10-day time course. Maximal stimulation of O₂ consumption occurred at day three, at which point the capacity for peroxidase-mediated O₂ consumption was three-to four-fold higher than that of the control O₂ consumption rate. Peroxidase-mediated O₂ consumption was sensitive to inhibition by 50 m M ascorbate and by cyanide. Cyanide titration curves indicated that O₂ consumption by peroxidase was much more sensitive to inhibition by cyanide than was O₂ consumption by cytochrome oxidase (I₅₀ < 1.6 μ M and I₅₀= 18.3 μ M cyanide, respectively). By using evidence from a combination of cyanide titration curves and ascorbate inhibition, we concluded that despite a large capacity for peroxidase-mediated O₂ consumption, peroxidase did not measurably contribute to control rates of O₂ consumption. In the absence of effectors, O₂ consumption was mediated primarily by cytochrome oxidase.     

Hydrogen uptake in Anabaena sp. strain CA does not protect nitrogenase from inactivation by hyperbaric oxygen

Abstract Two mutants of Anabaena sp. strain CA were used to demonstrate that oxygen-dependent hydrogen uptake was not the primary means to protect the nitrogenase enzyme complex from the deleterious effects of hyperbaric oxygen in vivo. Exposure to air caused the immediate and irreversible inactivation of nitrogenase activity in an oxygen-sensitive mutant, designated strain 22Y. Inactivation was concomitant with the destruction of the molybdo-iron (MoFe) protein of the nitrogenase complex. The mutant 22Y expressed an O₂-stable, Ni²⁺-stimulated hydrogen uptake of up to 2.7 μM H₂ per mg dry wt per h. Conversely, after exposure to 1% CO₂-99% O₂ for 3 h, both wild-type strain CA and a hydrogen uptake deficient (Hup⁻) mutant, strain N9AR, recovered 70–80% of their original acetylene reduction capacity with no apparent perturbations in the MoFe protein.     

EXTRACELLULAR PEROXIDASE-MEDIATED OXYGEN CONSUMPTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

The unicellular green alga Chlamydomonas reinhardtii Dang. displays a high capacity for salicylhydroxamic acid (SHAM)—stimulated O₂ consumption, mediated by extracellular peroxidaie. Addition of exogenous NADH also resulted in stimulation of O₂ consumption. The SHAM-and NADH-stimulated peroxidase activity was partially sensitive to inhibition by exogenous superoxide dismutase, ascorbate, and gentisic acid. These compounds did not inhibit O₂ consumption in the absence of effectors. SHAM-and NADH-stimulated peroxidase activity also was sensitive to inhibition by cyanide, and cyanide titration curves indicated that O₂ consumption by peroxidase was more cyanide-sensitive than O₂ consumption by cytochrome oxidase. The differential sensitivity to cyanide was used to estimate partitioning of O₂ consumption between mitochondrial respiration and extracellular peroxidase. We suggest that, despite a large capacity for peroxidase-me-diated O₂ consumption, peroxidase did not consume O₂ at detectable rates in the absence of effectors. Therefore, in the absence of effectors, measured rates of O₂ consumption represented the rate of mitochondrial respiration .     

Alteration of N nutrition in Myrica gale induces changes in nodule growth, nodule activity and amino acid composition

Changes in nodule growth and activity and in the concentrations of soluble N compounds in nodules, leaves and xylem sap under conditions of altered N nutrition in the actinorhizal plant Myrica gale L. are reported. Altering the N nutrition of symbiotic plants may alter the internal regulation of combined N which in turn may regulate nodule growth and activity. Flushing nodules daily with 100% O₂ caused a decline in amide concentration and an increase in nodule growth although plants had recovered some nitrogenase activity within 4 h of exposure to O₂. Samples of nodules, leaves and xylem sap were derivatized and amino acids identified and quantified using either reverse phase high performance liquid chromatography or gas chromatography-mass spectrometry in single ion monitoring mode. The ratio of asparagine in the nodules to that in the xylem was much higher in plants fed N (6.7 for NH⁺₄-fed and 8.3 for NO⁻₃-fed plants) than for N₂-fixing plants (2.5). Significant amounts of ¹⁵N added as ¹⁵NH⁺₄ or ¹⁵NO⁻₃ accumulated in nodules following accumulation in the shoot which is consistent with the translocation of N to the nodules via the phloem. The uptake of ¹⁵NH⁺₄ led to the synthesis and subsequent translocation of glutamine in the xylem sap. These results are discussed in terms of the feedback mechanisms that may regulate nitrogen fixation in Myrica root nodules.     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species

Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O₂ (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO₃⁻ and O₂ and, under controlled O₂ supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N₂O:N₂). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.     

Azide-stimulated oxygen consumption by the green alga Selenastrum minutum

Dark O₂ consumption by the green alga Selenastrum minutum was sensitive to inhibition by the cytochrome pathway respiration inhibitor cyanide in the absence of an alternative oxidase inhibitor, consistent with previous work that suggested that this alga lacks alternative oxidase capacity. In contrast, addition of low concentrations of the cytochrome pathway inhibitor azide (50–750 μ M ) resulted in a stimulation of dark O₂ consumption, while higher concentrations of azide (1–2 m M ) partially inhibited O₂ consumption. Measurements of changes in cellular levels of pyruvate, malate and pyridine nucleotides upon cyanide addition were consistent with the absence of alternative oxidase capacity, and suggested that cyanide inhibition of O₂ consumption was not due to nonspecific effects of cyanide. Addition of salicylhydroxamic acid (SHAM) also resulted in an increase in the rate of O₂ consumption. Both azide- and SHAM-stimulated O₂ consumption were sensitive to inhibition by 50 m M ascorbate or by cyanide. However, the ubiquinone analogs chloroquine and quinacrine specifically inhibited azide-stimulated O₂ consumption, with only minor effects on SHAM-stimulated O₂ consumption. These results suggest that azide-stimulated O₂ consumption was not mediated by the previously characterized SHAM-stimulated oxidase, and are consistent with the possibility that azide-stimulated O₂ consumption is mediated by a plasma membrane redox system.