Relationship between Ureide N and N(2) Fixation, Aboveground N Accumulation, Acetylene Reduction, and Nodule Mass in Greenhouse and Field Studies with Glycine max L. (Merr)

The relationship between ureide N and N₂ fixation was evaluated in greenhouse-grown soybean (Glycine max L. Merr.) and lima bean (Phaseolus lunatus L.) and in field studies with soybean. In the greenhouse, plant N accumulation from N₂ fixation in soybean and lima bean correlated with ureide N. In soybean, N₂ fixation, ureide N, acetylene reduction, and nodule mass were correlated when N₂ fixation was inhibited by applying KNO₃ solutions to the plants. The ureide-N concentrations of different plant tissues and of total plant ureide N varied according to the effectiveness of the strain of Bradyrhizobium japonicum used to inoculate plants. The ureide-N concentrations in the different plant tissues correlated with N₂ fixation. Ureide N determinations in field studies with soybean correlated with N₂ fixation, aboveground N accumulation, nodule weight, and acetylene reduction. N₂ fixation was estimated by ¹⁵N isotope dilution with nine and ten soybean genotypes in 1979 and 1980, respectively, at the V9, R2, and R5 growth stages. In 1981, we investigated the relationship between ureide N, aboveground N accumulation, acetylene reduction, and nodule mass using four soybean genotypes harvested at the V4, V6, R2, R4, R5, and R6 growth stages. Ureide N concentrations of young stem tissues or plants or aboveground ureide N content of the four soybean genotypes varied throughout growth correlating with acetylene reduction, nodule mass, and aboveground N accumulation. The ureide-N concentrations of young stem tissues or plants or aboveground ureide-N content in three soybean genotypes varied across inoculation treatments of 14 and 13 strains of Bradyrhizobium japonicum in 1981 and 1982, respectively, and correlated with nodule mass and acetylene reduction. In the greenhouse, results correlating nodule mass with N₂ fixation and ureide N across strains were variable. Acetylene reduction in soybean across host-strain combinations did not correlate with N₂ fixation and ureide N. N₂ fixation, ureide N, acetylene reduction, and nodule mass correlated across inoculation treatments with strains of Bradyrhizobium spp. varying in effectiveness on lima beans. Our data indicate that ureide-N determinations may be used as an additional method to acetylene reduction in studies of the physiology of N₂ fixation in soybean. Ureide-N measurements also may be useful to rank strains of B. japonicum for effectiveness of N₂ fixation.     

Hydrazine as a substrate and inhibitor of Azotobacter vinelandii nitrogenase

Hydrazine has been tested as a substrate and inhibitor of nitrogenase from Azotobacter vinelandii. It is a linear noncompetitive inhibitor of acetylene reduction, with K_il = K_is = 80 mM at pH 8.0. Carbon monoxide is a linear noncompetitive inhibitor of hydrazine reduction with K_ii = K_is = 2 × 10^?4atm. The inhibition of acetylene reduction by hydrazine is unaffected by the presence of hydrogen, and hydrogen does not inhibit the reduction of hydrazine. Hydrazine can completely suppress hydrogen evolution, while not inhibiting phosphate hydrolysis. The apparent K_m for hydrazine reduction varies with pH, reaching a limiting value of about 25 mM at high pH. The apparent K_i for hydrazine inhibition of hydrogen evolution reaches a similar limiting value at high pH. By varying the concentration of ATP it is possible to alter the relative allocation of electrons to acetylene or hydrazine. Hydrazine is a relatively more potent inhibitor of acetylene reduction at low levels of ATP. It is concluded that hydrazine is able to react effectively with a less reduced state of the enzyme from A. vinelandii than is acetylene or dinitrogen.     

Pyridine nucleotides and H2 as electron donors to the respiratory and photosynthetic electron-transfer chains and to nitrogenase in Anabaena heterocysts

The pathways through which NADPH, NADH and H₂ provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O₂ uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H₂ supported cyanide-sensitive, respiratory O₂ uptake and light-enhanced, cyanide-insensitive uptake of O₂ resulting from electron donation to O₂ at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O₂ or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H₂ supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H₂ as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented.     

The Relationship between H(2) Evolution and Acetylene Reduction in Pisum sativum-Rhizobium leguminosarum Symbioses Differing in Uptake Hydrogenase Activity

Peas (Pisum sativum L.) were inoculated with strains of Rhizobium leguminosarum having different levels of uptake hydrogenase (Hup) activity and were grown in sterile Leonard jars under controlled conditions. Rates of H₂ evolution and acetylene reduction were determined for intact nodulated roots at intervals after the onset of darkness or after removal of the shoots. Hup activity was estimated using treatment plants or equivalent plants from the growth chamber, by measuring the uptake of H₂ or ³H₂ in the presence of acetylene. In all cases, the rate of H₂ evolution was a continuous function of the rate of acetylene reduction. In symbioses with no demonstrable Hup activity, H₂ evolution increased in direct proportion to acetylene reduction and the slopes were similar with the Hup⁻ strains NA502 and 128C79. Hup activity was similar in strains 128C30 and 128C52 but significantly lower in strain 128C54. With these strains, the slopes of the H₂ evolution versus acetylene reduction curves initially increased with acetylene reduction, but became constant and similar to those for the Hup⁻ strains at high rates of acetylene reduction. On these parallel portions of the curves, the decreases in H₂ evolution by Hup⁺ strains were similar in magnitude to their H₂-saturated rates of Hup activity. The curvilinear relationship between H₂ evolution and acetylene reduction for a representative Hup⁺ strain (128C52) was the same, regardless of the experimental conditions used to vary the nitrogenase activity.     

Localized Tetrazolium Reduction in Relation to N2 Fixation, CO2 Fixation, and H2 Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N₂ fixation (acetylene reduction), ¹⁴CO₂ fixation, and ³H₂ utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, ¹⁴CO₂ fixation and ³H₂ utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N₂-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.     

Limitation of acetylene reduction (nitrogen fixation) by photosynthesis in soybean having low water potentials

The role of photosynthesis and transpiration in the desiccation-induced inhibition of acetylene reduction (nitrogen fixation) was investigated in soybean (Glycine max [L.] Merr. var. Beeson) using an apparatus that permitted simultaneous measurements of acetylene reduction, net photosynthesis, and transpiration. The inhibition of acetylene reduction caused by low water potentials and their aftereffects could be reproduced by depriving shoots of atmospheric CO₂ even though the soil remained at water potentials that should have favored rapid acetylene reduction. The inhibition of acetylene reduction at low water potentials could be partially reversed by exposing the shoots to high CO₂ concentrations. When transpiration was varied independently of photosynthesis and dark respiration in plants having high water potentials, no effects on acetylene reduction could be observed. There was no correlation between transpiration and acetylene reduction in the CO₂ experiments. Therefore, the correlation that was observed between transpiration and acetylene reduction during desiccation was fortuitous. We conclude that the inhibition of shoot photosynthesis accounted for the inhibition of nodule acetylene reduction at low water potentials.     

Effect of temperature on h(2) evolution and acetylene reduction in pea nodules and in isolated bacteroids

Nitrogenase (EC 1.7.99.2) activity in pea (Pisum savitum) nodules formed after infection with Rhizobium leguminosarum (lacking uptake hydrogenase) was measured as acetylene reduction, H₂ evolution in air and H₂ evolution in Ar:O₂. With detached roots the relative efficiency, calculated from acetylene reduction, showed a decrease (from 55 to below 0%) with increasing temperature. With excised nodules and isolated bacteroids similar results were obtained. However, the relative efficiency calculated from H₂ evolution in Ar:O₂ was unaffected by temperature. Measurements on both excised nodules and isolated bacteroids showed a marked difference between acetylene reduction and H₂ evolution in Ar:O₂ with increased temperature, indicating that either acetylene reduction or H₂ evolution in Ar:O₂ are inadequate measures of nitrogenase activity at higher temperature.     

Acetylene reduction (nitrogen fixation) and metabolic activities of soybean having various leaf and nodule water potentials

An apparatus was designed that permitted acetylene reduction (N₂ fixation) by root nodules to be measured in situ simultaneously with net photosynthesis, dark respiration, and transpiration of the shoot in soybean plants (Glycine max [L.] Merr. var. Beeson). Tests showed that acetylene reduction was linear with time for at least 5 hours, except for the first 30 to 60 minutes. Endogenous ethylene production did not affect the measurements. Successive determinations of acetylene reduction could be made without apparent aftereffects on the plant.     

Nitrogen Fixation Associated with Rinsed Roots and Rhizomes of the Eelgrass Zostera marina

Nitrogen fixation was associated with the rinsed roots and rhizomes of the seagrass, Zostera marina L. Nitrogenase activity (acetylene reduction) was greater on rhizomes compared to roots, and on older roots and rhizomes relative to younger tissue. Compared to aerobic assays, anaerobic or microaerobic conditions enhanced the rate of acetylene reduction by rhizomes with attached roots, with the highest activity (100 nanomoles per gram dry weight per hour) occurring at pO₂ = 0.01 atmosphere. Addition of glucose, sucrose, or succinate also increased the rate of acetylene reduction under anaerobic conditions, with glucose providing the most stimulation. In one experiment, comparison of acetylene reduction assays with ¹⁵N₂ incorporation yielded a ratio of about 2.6:1. Seagrass communities are thought to be limited by the availability of nitrogen and, therefore, nitrogenase activity directly associated with their roots and rhizomes suggests the possibility of a N₂-fixing flora which may subsidize their nutritional demand for nitrogen.     

Diffusion of Gases through Plant Tissues : Entry of Acetylene into Legume Nodules

I have measured acetylene diffusion through plant tissues including nodules from several species of legume—vetch, peas, soybeans, and Sesbania rostrata. The observed half-time for reequilibration of internal and external concentration is less than 1 minute for typical nodules. Inward diffusion of acetylene in air is rapid relative to the use of acetylene by nitrogenase so that diffusion of acetylene would not be a significant limiting factor for nitrogenase activity in air. However, under an atmosphere of Ar:O₂ where there is no N₂ reduction, the inward diffusion rate of acetylene into larger nodules could produce a measurable limitation of observed nitrogenase activity at low acetylene concentrations.     

Quantification and Removal of Some Contaminating Gases from Acetylene Used to Study Gas-Utilizing Enzymes and Microorganisms

Acetylene generated from various grades of calcium carbide and obtained from commercial- and purified-grade acetylene cylinders was shown to contain high concentrations of various contaminants. Dependent on the source of acetylene, these included, at maximal values, H₂ (0.023%), O₂ (0.779%), N₂ (3.78%), PH₃ (0.06%), CH₄ (0.073%), and acetone (1 to 10%). The concentration of the contaminants in cylinder acetylene was highly dependent on the extent of cylinder discharge. Several conventional methods used to partially purify cylinder acetylene were compared. A small-scale method for extensively purifying acetylene is described. An effect of acetylene quality on acetylene reduction assays conducted with purified nitrogenase from Azotobacter vinelandii was demonstrated.     

Biological Dinitrogen Fixation (Acetylene Reduction) Associated with Florida Mangroves

Biological dinitrogen fixation in mangrove communities of the Tampa Bay region of South Florida was investigated using the acetylene reduction technique. Low rates of acetylene reduction (0.01 to 1.84 nmol of C₂H₄/g [wet weight] per h) were associated with plant-free sediments, while plant-associated sediments gave rise to slightly higher rates. Activity in sediments increased greatly upon the addition of various carbon sources, indicating an energy limitation for nitrogenase (C₂H₂) activity. In situ determinations of dinitrogen fixation in sediments also indicated low rates and exhibited a similar response to glucose amendment. Litter from the green macroalga, Ulva spp., mangrove leaves, and sea grass also gave rise to significant rates of acetylene reduction.     

Acetylene, Not Ethylene, Inactivates the Uptake Hydrogenase of Actinorhizal Nodules during Acetylene Reduction Assays

Acetylene reduction assays were shown to inactivate uptake hydrogenase activity to different extents in one Casuarina and two Alnus symbioses. Inactivation was found to be caused by C₂H₂ and not by C₂H₄. Acetylene completely inactivated the hydrogenase activity of intact root systems of Alnus incana inoculated with Frankia strain Avcl1 in 90 minutes, as shown by a drop in the relative efficiency of nitrogenase from 1.0 to 0.73. The hydrogenase of Frankia preparations (containing vesicles) and of cell-free extracts (not containing vesicles) from the same symbiosis was much more susceptible to acetylene inactivation. Cell-free extracts lost all hydrogenase activity after 5 minutes of exposure to acetylene. The hydrogenase activity of intact root systems of Casuarina obesa was less sensitive to acetylene than that of root systems of A. incana, since the relative efficiency of nitrogenase changed only from 1.0 to 0.95 over 90 minutes. Frankia preparations and cell-free extracts of C. obesa still retained hydrogenase activity after a 10 minute-exposure to acetylene.     

Nitrogen metabolism of soybeans: I. Effect of tungstate on nitrate utilization, nodulation, and growth

The effects of N source (6 mm nitrogen as NO₃⁻ or urea) and tungstate (0, 100, 200, 300, and 400 μm Na₂ WO₄) on nitrate metabolism, nodulation, and growth of soybean (Glycine max [L.] Merr.) plants were evaluated. Nitrate reductase activity and, to a lesser extent, NO₃⁻ content of leaf tissue decreased with the addition of tungstate to the nutrient growth medium. Concomitantly, nodule mass and acetylene reduction activity of NO₃⁻-grown plants increased with addition of tungstate to the nutrient solution. In contrast, nodule mass and acetylene reduction activity of urea-grown plants decreased with increased nutrient tungstate levels. The acetylene reduction activity of nodulated roots of NO₃⁻-grown plants was less than 10% of the activity of nodulated roots of urea-grown plants when no tungstate was added. At 300 and 400 μm tungstate levels, acetylene reduction activity of nodulated roots of NO₃⁻-grown plants exceeded the activity of comparable urea-grown plants.     

Factors affecting the reduction of acetylene by Rhizobium-soybean cell associations in vitro

The acetylene reduction assay was used to measure presumed N₂-reducing activity in Rhizobium-soybean cell associations in vitro. No acetylene reduction was observed in liquid suspensions of these organisms, but cells plated onto an agar medium from a liquid suspension of Rhizobium and soybean cells exhibited acetylene-dependent production of ethylene after 7 to 14 days. Aggregates of soybean cells 0.5 to 2.0 mm in diameter were required for this activity. Decreasing oxygen from 0.20 atm to 0.10, 0.04, or 0.00 atm completely inhibited acetylene reduction. The presence of 2,4-dichlorophenoxyacetic acid or kinetin increased endogenous ethylene production and inhibited acetylene-dependent ethylene production. Acetylene reduction was observed with three out of four strains of R. japonicum tested, and three rhizobial strains, which produce root nodules on cowpeas but not soybeans, formed an association capable of acetylene-dependent ethylene production.     

The Azolla-Anabaena azollae relationship

The heterocystous blue-green alga, Anabaena azollae, was isolated from the leaf cavities of the water fern, Azolla caroliniana, where it occurs as an endophyte. The isolated alga was capable of light dependent CO₂ fixation and acetylene reduction. Aerobic dark acetylene reduction occurred and was dependent upon endogenous substrates. Vegetative cells of the alga reduced nitro-blue tetrazolium chloride (NBT) to blue formazan. Heterocysts did not. Heterocysts reduced triphenyl tetrazolium chloride (TTC) to red formazan faster than vegetative cells. Reduction of TTC by both heterocysts and vegetative cells was much more rapid than has been reported for free-living heterocystous blue-green algae. Both NBT and TTC inhibited acetylene reduction and CO₂ fixation. The inhibition by TTC was more closely correlated to the time of exposure of the cells to the reagent and to the amount of deposition per cell than to the number of cells containing red formazan. No differential inhibition of acetylene reduction versus CO₂ fixation was observed. Autoradiography showed that CO₂ fixation occurred only in vegetative cells. Heterocysts caused a darkening of nuclear emulsions (chemography). This observation has been employed by others as an index of reducing activity in these cells. DCMU inhibited the acetylene reducing capacity of alga isolated from dark pretreated fronds more rapidly and to a greater extent than that in alga isolated from light pretreated fronds. Ammonia in excess of 5 mM was required before any inhibition of acetylene reduction was observed under either aerobic or anaerobic conditions in the light.     

Diurnal variation in algal acetylene reduction (nitrogen fixation) in situ

Diurnal variation in algal nitrogen fixation was studied in Lake Mendota, Wisconsin, during the summers of 1971 to 1973. Approximately two-thirds of the daily acetylene reduction in the surface decimeter occurred before noon. The decline in acetylene reduction (nmoles/liter·hr) near midday was partially because the algae relocated themselves at greater depths. However, acetylene reducing activity (nmoles per A₆₆₃ unit chlorophyll a per hour) also decreased as midday approached. Occasionally algae would resurface near the end of the day. On average, acetylene reduction (nmoles per liter per hour) was maximum at about 0900 Central standard time in the top decimeter, and acetylene reduction between 0830 and 0930 Central standard time represented 13% of the total daily acetylene reduction. Furthermore, acetylene reduction in the top decimeter, on average, represented 3.6% of the total acetylene reduction in the column. Calculation of the contribution by nitrogen fixation to a lake's fixed nitrogen budget is discussed.     

Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria

Acetylene (0.1 atm) caused complete or almost complete inhibition of reduction of N₂O by whole cell suspensions of Pseudomonas perfectomarinus, P. aeruginosa and Micrococcus denitrificans. Acetylene did not inhibit reduction of NO₃^? or NO₂^? by these organisms. In the presence of acetylene there was stoichiometric conversion of NO₃^? or NO₂^? to N₂O with negligible subsequent reduction of the latter. In the absence of acetylene there was no or only transient accumulation of N₂O. The data are consistent with the view that N₂O is an obligatory intermediate in the reduction of NO₂^? to N₂ in all of the three organisms studied.     

Continuous, automated acetylene reduction assays using intact plants

An automated method was developed for continuous, in situ determination of acetylene reduction (N₂ fixation) by intact soybean plants (Glycine max [L.]). The culture vessel containing the roots of intact plants grown in sand culture is sealed at the surface and an air-acetylene mixture continuously injected into the root chamber. The effluent gas is automatically sampled and injected into a gas chromatograph. Continuous acetylene assay at intervals as short as 3.5 min may be made over a period of several days, without attention, except for plant watering. Adverse effects of prolonged exposure of the root system to acetylene were mitigated by pulse injection of acetylene for 20 min followed by 40 min of acetylene-free air. Bare root systems can be suspended in a reaction chamber and sprayed with water or nutrient solution; this permits periodic removal of the root system for sampling nodules.     

Enzymes of ammonia assimilation and ureide biosynthesis in soybean nodules: effect of nitrate

The effect of nitrate on N₂ fixation and the assimilation of fixed N₂ in legume nodules was investigated by supplying nitrate to well established soybean (Glycine max L. Merr. cv Bragg)-Rhizobium japonicum (strain 3I1b110) symbioses. Three different techniques, acetylene reduction, ¹⁵N₂ fixation and relative abundance of ureides ([ureides/(ureides + nitrate + α-amino nitrogen)] × 100) in xylem exudate, gave similar results for the effect of nitrate on N₂ fixation by nodulated roots. After 2 days of treatment with 10 millimolar nitrate, acetylene reduction by nodulated roots was inhibited by 48% but there was no effect on either acetylene reduction by isolated bacteroids or in vitro activity of nodule cytoplasmic glutamine synthetase, glutamine oxoglutarate aminotransferase, xanthine dehydrogenase, uricase, or allantoinase. After 7 days, acetylene reduction by isolated bacteroids was almost completely inhibited but, except for glutamine oxoglutarate aminotransferase, there was still no effect on the nodule cytoplasmic enzymes. It was concluded that, when nitrate is supplied to an established symbiosis, inhibition of nodulated root N₂ fixation precedes the loss of the potential of bacteroids to fix N₂. This in turn precedes the loss of the potential of nodules to assimilate fixed N₂.