Immediate acetylene reduction by excised grass roots not previously preincubated at low oxygen tensions

Excised roots of Spartina alterniflora Loisel. and corn reduced acetylene in air without the previously reported period of zero activity lasting 8 to 18 hours. The profiles of acetylene-dependent ethylene accumulation by excised roots and intact plants of S. alterniflora were similar. No significant change in the number of bacteria associated with the roots was detectable during the assay. Most of the nitrogenase activity was detected in the roots and rhizomes of the plants. The salt marsh sediment also was capable of reducing acetylene. Additional damage to roots by washing and cutting increased the rate of acetylene reduction with samples incubated in air. Low concentrations of nitrate significantly inhibited the nitrogenase activity associated with the sediment and excised roots, but not with intact plants. Rates of acetylene reduction by excised corn roots were low. Oxidation and endogenous production of ethylene in the absence of acetylene were negligible. Measurements made with excised grass roots as described probably reflect the occurrence and magnitude of nitrogenase activity associated with the plants in the field.     

Nitrogenase activity in the papyrus swamps of Lake Naivasha,Kenya

Nitrogenase activity (acetylene reduction activity) was found to occur universally in the Cyperus papyrus swamp in Lake Naivasha. Low rates of acetylene reduction activity (0.9–104.9 nmol C₂H₄ g d.wt. roots^-1 h^-1) were associated with excised roots of C. papyrus but higher rates of activity (89.0–280.4 nmol C₂H₄ g d.wt. roots^-1 h^-1) were associated with intact root systems of the plant. It was estimated that nitrogen fixation associated with young roots alone could supply about 26% of the nitrogen requirements of growing papyrus plants. Acetylene reduction activity in the lake bottom sediments was generally low and associated with adjacent papyrus stands. Plate counts of putative aerobic and facultatively anaerobic N₂-fixing bacteria associated with papyrus roots showed the presence of high numbers of diazotrophs (5.4 × 10⁶ CFU g d.wt. roots^-1). Fewer numbers of N₂-fixing bacteria were detected in the sediments (1.9 × 10³-3.2 × 10⁴ CFU g d.wt. sediment^-1).     

Nitrogen fixation in the rhizosphere and rhizoplane of barley

Summary Aerobic and anaerobic N₂-fixing bacteria developed in the rhizosphere of barley seedlings and exhibited N₂ase activity when seedlings were grown in sterilized sand-nutrient cultures containing low levels of combined nitrogen. The source of the N₂-fixing bacteria appeared to be the seed. Average daily rates up to 0.9 μmoles C₂H₄ h⁻¹ g⁻¹ dry root tissue were measured, but the intensity of the activity was affected by moisture levels and concentration of combined N in the rhizosphere. Removal and washing of the roots did not remove the activity, and roots remained active even after surface-sterilization. An unidentified aerobic N₂-fixing bacterium was isolated from the rhizoplane of active barley roots. Inoculation of barley seedlings with the aerobic N₂-fixing bacterium enhanced N₂ase activity of excised roots 10-fold, with average rates of 0.9, 1.1 and 1.3 μmoles h⁻¹ g⁻¹ dry root assayed under pO₂ of 0.01, 0.02 and 0.04 atm respectively. The aerobic N₂-fixing bacterium also exhibited N₂ase activity when inoculated into the rhizosphere of oat, rice and wheat seedlings. Microscopic observations of sterilized live and stained barley roots suggest that the aerobic N₂-fixing bacterium is an endophyte which infects root tissue and metamorphoses into vesicle-like structures.     

Ecology of heterotrophic dinitrogen fixation in the rhizosphere of mangrove plant community at the Ganges river estuary in India

Summary Heterotrophic dinitrogen fixation in root associations of successional stages of the tropical mangrove plant community at the Ganges river estuary in India was investigated by excised-root acetylene reduction assay, and enumeration and identification of diazotrophic bacteria from sediment, root and tidal water samples. High to very high rates of nitrogenase activity (64–130 nmol C₂H₄/g dry root/h) were associated with washed excised roots of seven common early-successional mangrove species at the inundated swamps. Declining, late-successional mangroves at the occasionally inundated ridges had considerably lower values and the “declined” mangroves and other non-littoral species at embankment protected highlands had very low to insignificant values of root nitrogenase activity. Total and inorganic nitrogen contents of the mangrove sediments were low and were positively related to the stages of physiographic succession. Plant-associated sediments of particularly the old formation swamps had very high C/N ratios. Nine isolates of nitrogen-fixing bacteria belonging to all known O₂ response groups were distinguished from a large population of diazotrophs associated with roots of mangroves and other associate plant species of the community. The isolates differed with respect to their N₂-fixation efficiency and halotolerance in pure culture. There was no specificity of any of the bacterial isolates to any of the plant species of the community but a higher number of efficient isolates were seen to be associated with mangroves at the swampy succession. Sediment-free tidal water also contained a large population of microaerophilic and anaerobic N₂-fixing bacteria.     

Enumeration of Free-Living Aerobic N2-Fixing H2-Oxidizing Bacteria by Using a Heterotrophic Semisolid Medium and Most-Probable-Number Technique

A heterotrophic semisolid medium was used with two sensitive assay methods, C₂H₂ reduction and O₂-dependent tritium uptake, to determine nitrogenase and hydrogenase activities, respectively. Organisms known to be positive for both activities showed hydrogenase activity in both the presence and absence of 1% C₂H₂, and thus, it was possible to test a single culture for both activities. Hydrogen uptake activity was detected for the first time in N₂-fixing strains of Pseudomonas stutzeri. The method was then applied to the most-probable-number method of counting N₂-fixing and H₂-oxidizing bacteria in some natural systems. The numbers of H₂-oxidizing diazotrophs were considerably higher in soil surrounding nodules of white beans than they were in the other systems tested. This observation is consistent with reports that the rhizosphere may be an important ecological niche for H₂ transformation.     

Comparing Time Course Profiles of Immediate Acetylene Reduction by Grasses and Legumes

The time course profiles of C₂H₂ reduction by intact Scirpus olneyi (bulrush), Oryza sativa (rice) and Spartina alterniflora (cordgrass) with roots in atmospheres of N₂ and 30-day-old Glycine max (soybean) in air were all immediately linear. This is the first report of immediately linear rates of C₂H₂ reduction by grass roots removed from soil. The immediately linear profile of C₂H₂ reduction by soil-free grass roots was achieved by preventing contact between the roots and air. Roots of soybeans and S. olneyi receiving pretreatments of O₂ above normal environmental levels for 15 min before assay exhibited a short delay in C₂H₂ reduction. These initially nonlinear rates of C₂H₂ reduction are attributable to transient O₂ inhibition of nitrogenase. Initial nonlinear rates of C₂H₂ reduction were also observed with immature soybean plants and with intact plant assays of O. sativa and S. olneyi in which C₂H₂ was injected into cylinders surrounding the plant tops. These results indicate that, apart from O₂ inhibition of nitrogenase, the diffusion of C₂H₂ and C₂H₄ between the nitrogen-fixing sites and the sampling ports may cause initial nonlinear rates of C₂H₂ reduction. We conclude that in situ plant-associated nitrogenase activity should result in immediate reduction of C₂H₂ and that linear rates are observed when the proper assay conditions are used. Our data suggest that nitrogen fixation is closely associated with the roots of S. olneyi, O. sativa, and S. alterniflora growing in salt marsh sediment.     

Adaptation of Nitrogen Fixation by Intact Soybean Nodules to Altered Rhizosphere pO(2)

The N₂-fixing legume nodule requires O₂ for ATP production; however, the O₂ sensitivity of nitrogenase dictates a requirement for a low pO₂ inside the nodule. The effects of long term exposures to various pO₂s on N₂[C₂H₂] fixation were evaluated with intact soybean (Glycine max [L.] Merr., var. Wye) plants. Continuous exposure of their rhizosphere to a pO₂ of 0.06 atmospheres initially reduced nitrogenase activity by 37 to 45% with restoration of original activity in 4 to 24 hours and with no further change in tests up to 95 hours; continuous exposure to 0.02 atmosphere of O₂ initially reduced nitrogenase activity 72%, with only partial recovery by 95 hours. Similar exposures to a pO₂ of 0.32 atmospheres had little effect on N₂[C₂H₂] fixation; a pO₂ of 0.89 atmospheres initially reduced nitrogenase activity by 98% with restoration to only 14 to 24% of that of the ambient O₂ controls by 95 hours. Re-exposure to ambient pO₂ of plants adapted to nonambient pO₂s reduced N₂[C₂H₂] fixation to similar magnitudes as the reductions which occurred upon initial exposure to variant pO₂ conditions, and a time period was required to readapt to ambient O₂. It is concluded that the N₂[C₂H₂]-fixing system of intact soybean plants is able to adapt to a wide range of external pO₂s as probably occur in soil. We postulate that this occurs through an undefined mechanism which enables the nodule to maintain an internal pO₂ optimal for nitrogenase activity.     

Interaction of Combined Nitrogen with the Expression of Root-Associated Nitrogenase Activity in Grasses and with the Development of N(2) Fixation in Soybean (Glycine max L. Merr.)

Soluble root N concentrations of corn, sorghum, pearl millet, rice, wild rice, and soybeans were determined and related to measurements of nitrogenase activity and changes in availability of combined N to plants. In corn, sorghum, and pearl millet, applications of fertilizer N increased soluble root N concentrations, but root-associated nitrogenase activity was negligible in control and treated plants. Applications of NH₄NO₃ to rice increased the water soluble root N concentrations and inhibited root-associated nitrogenase activity. In wild rice, root-associated nitrogenase activity was absent during vegetative growth and developed at the reproductive growth stage. The soluble root N concentration decreased progressively as wild rice grew indicating that the availability of combined N in the root environment declined. Therefore, development of nitrogenase activity in wild rice is associated with the change in availability of combined N in the root environment. The development of nitrogenase activity in wild rice was probably not due to colonization of roots by N₂-fixing bacteria because most probable numbers of recovery did not significantly vary throughout the plants' growth cycle. In field-grown soybeans with or without fertilizer N application, we also observed a relationship between a decrease in soluble root N concentration and the development of nitrogenase activity.     

Enumeration and Localization of N2-Fixing Bacteria Associated with Roots of Spartina alterniflora Loisel

Numbers and possible locations of N₂-fixing bacteria were investigated in roots of Spartina alterniflora Loisel, which support nitrogenase activity in the undisturbed native habitat. N₂-fixing bacteria were recovered in cultures both from S. alterniflora roots and from the surrounding sediment, and they formed a greater proportion of the bacteria recovered from root homogenates than from salt-marsh sediment. N₂-fixing bacteria were recovered in high numbers from the rhizoplane of S. alterniflora after roots were treated with 1 or 5% chloramine-T for 1 h or with 1% NaOCl for 1 or 2 h. Immersing S. alterniflora roots in 5% NaOCl for 1 h was more effective in distinguishing bacteria inside the roots since this treatment nearly eliminated N₂-fixing bacteria recoverable from the rhizoplane, although high numbers of N₂-fixing bacteria were recovered from homogenates of roots treated with 5% NaOCl for 1 h. However, this treatment was less effective with roots of Zea mays L. (Funks G4646) and Sorghum bicolor (L.) Moench (CK-60 A), indicating that techniques to surface sterilize roots should be evaluated for different plants. Bacteria were observed by light and electron microscopy inter- and intracellularly in the cortex and in the aerenchyma of S. alterniflora roots. This study clearly shows that bacteria, including N₂ fixers, colonize the interior of roots of S. alterniflora growing in a Chesapeake Bay, Maryland, salt marsh.     

Isolation and Characterization of Dinitrogen-Fixing Bacteria from the Rhizosphere of Temperate Cereals and Forage Grasses

N₂(C₂H₂)-fixing bacteria were isolated from the rhizosphere of various cereals and forage grasses grown in the greenhouse and from the rhizosphere of field-grown wheat in Sweden. All 46 isolates from the greenhouse plants lost their nitrogenase activity during purification. By imposing a stronger selection pressure, we obtained pure isolates with nitrogenase activity from field-grown wheat. Some isolates were identified as Enterobacter agglomerans and Bacillus polymyxa, but several bacteria of uncertain taxonomy also occurred. One of the isolates grew and reduced acetylene only in the presence of other bacteria or certain vitamins. Species of Azotobacter or Azospirillum could not be isolated from the rhizosphere of any of the cereals and forage grasses studied.     

Hydrogen-Dependent Nitrogenase Activity and ATP Formation in Rhizobium japonicum Bacteroids

Rhizobium japonicum 122 DES bacteroids from soybean nodules possess an active H₂-oxidizing system that recycles all of the H₂ lost through nitrogenase-dependent H₂ evolution. The addition of 72 μM H₂ to suspensions of bacteroids increased O₂ uptake 300% and the rate of C₂H₂ reduction 300 to 500%. The optimal partial pressure of O₂ was increased, and the partial pressure of O₂ range for C₂H₂ reduction was extended by adding H₂. A supply of succinate to bacteroids resulted in effects similar to those obtained by adding H₂. Both H₂ and succinate provided respiratory protection for the N₂-fixing system in bacteroids. The oxidation of H₂ by bacteroids increased the steady-state pool of ATP by 20 to 40%. In the presence of 50 mM iodoacetate, which caused much greater inhibition of endogenous respiration than of H₂ oxidation, the addition of H₂ increased the steady-state pool of ATP in bacteroids by 500%. Inhibitor evidence and an absolute requirement for O₂ indicated that the H₂-stimulated ATP synthesis occurred through oxidative phosphorylation. In the presence of 50 mM iodoacetate, H₂-dependent ATP synthesis occurred at a rate sufficient to support nitrogenase activity. The addition of H₂ to H₂ uptake-negative strains of R. japonicum had no effect on ATP formation or C₂H₂ reduction. It is concluded that the H₂-oxidizing system in H₂ uptake-positive bacteroids benefits the N₂-fixing process by providing respiratory protection of the O₂-labile nitrogenase proteins and generating ATP to support maximal rates of C₂H₂ reduction by oxidation of the H₂ produced from the nitrogenase system.     

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) by Pulp and Paper Mill Effluents and by Klebsiella Isolated from Effluents and Environmental Situations

High rates of acetylene (C₂H₂) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C₂H₄ produced per ml per h) and under anaerobic (up to 135 nmol of C₂H₄ produced per ml per h) conditions. Pasteurized effluent developed C₂H₂ reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N₂-fixing Azotobacter and Klebsiella were abundant, and N₂-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability.     

The acetylene-ethylene assay for n(2) fixation: laboratory and field evaluation

The methodology, characteristics and application of the sensitive C₂H₂-C₂H₄ assay for N₂ fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N₂ase-catalyzed reduction of C₂H₂ to C₂H₄, gas chromatographic isolation of C₂H₂ and C₂H₄, and quantitative measurement with a H₂-flame analyzer. As little as 1 μμmole C₂H₄ can be detected, providing a sensitivity 10³-fold greater than is possible with ¹⁵N analysis.

A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C₂H₂-reducing activity in the field. Applications to field samples included an evaluation of N₂ fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N₂ fixation rate of 30 to 33 kg N₂ fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N₂ fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N₂ fixing activity of soybeans. The validity of measuring N₂ fixation in terms of C₂H₂ reduction was established through extensive comparisons of these activities using defined systems, including purified N₂ase preparations and pure cultures of N₂-fixing bacteria.

With this assay it now becomes possible and practicable to conduct comprehensive surveys of N₂ fixation, to make detailed comparisons among different N₂-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N₂ fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N₂ fixation reaction.

     

Inoculation of forage grasses with N2-fixing Enterobacteriaceae

Summary Previous investigations indicated some forage grass roots in Texas are heavily colonized with N₂-fixing bacteria. The most numerous N₂-fixing bacteria were in the genera Klebsiella and Enterobacter. In the present investigation inoculation experiments were conducted using 18 isolates of these bacteria to determine if a N₂-fixing association could be established between the bacteria and the grassesCynodon dactylon andPanicum coloratum. Plants were grown in soil for approximately 5 months in a greenhouse and were measured periodically for dry matter, nitrogen accumulation, and acetylene reduction activity. Results of the investigation indicated that 25% of the plant-soil systems were active in acetylene reduction and the activity was high enough to indicate agronomically significant quantities of N₂ were being fixed (>8kg N ha⁻¹). However, plant systems extrapolated to fix>8 kg N ha⁻¹ contained less nitrogen and accumulated less dry matter than plants less active in acetylene reduction. Inocula could not be re-isolated from healthy grass roots indicating that the N₂-fixing activity may have not have been closely assiciated with plant roots. Future research is needed to determine factors limiting colonization of grass roots.     

Nitrogenase Activity Measurements in Intact Plants of Alnus incana

A technique for C₂H₂-reduction assay on intact plants of Alnus incana (L.) Moench was evaluated. Cloned plants were grown, in pots, on fine gravel. During assay only the pot was inserted into a Perspex incubation chamber of simple construction. The incubation volume was rather small, plants with various shoot heights could be used, and the shoot was not exposed to the C₂H₄ produced. Intact plants showed high and constant C₂H₂-reduction rates during several hours of incubation. In comparison, excised nodulated roots conventionally incubated in test tubes showed low and decreasing rates, due to removal of the photo-synthesizing shoot and injury to the root nodules when drawn from the pot. Repeated nitrogenase activity assays on the same intact individual plants did not affect growth. The technique thus proved useful in studies. where repeated nitrogenase activity measurements are important.     

Effect of localized nitrogen availability to soybean half-root systems on photosynthate partitioning to roots and nodules

Soybean (Glycine max [L.] Merr. cv Davis) was grown in a split-root growth system designed to maintain control of the root atmosphere. Two experiments were conducted to examine how 80% Ar:20% O₂ (Ar:O₂) and air (Air) atmospheres affected N assimilation (NH₄NO₃ and N₂ fixation) and the partitioning of photosynthate to roots and nodules. Application of NH₄NO₃ to nonnodulated half-root systems enhanced root growth and root respiration at the site of application. A second experiment applied Ar:O₂ or air to the two sides of nodulated soybean half-root systems for 11 days in the following combinations: (a) Air to both sides (Air/Air); (b) Air to one side, Ar:O₂ to the other (Air/Ar:O₂), and (c) Ar:O₂ to both sides (Ar:O₂/Ar:O₂). Results indicated that dry matter and current photosynthate (¹⁴C) were selectively partitioned to nodules and roots where N₂ was available. Both root and nodule growth on the Air side of Air/Ar:O₂ plants was significantly greater than the Ar:O₂ side. The relative partitioning of carbon and current photosynthate between roots and nodules on a half-root system was also affected by N₂ availability. The Ar:O₂ sides partitioned relatively more current photosynthate to roots (57%) than nodules (43%), while N₂-fixing root systems partitioned 36 and 64% of the carbon to roots and nodules, respectively. The Ar:O₂ atmosphere decreased root and nodule respiration by 80% and nitrogenase activity by 85% compared to half-root systems in Air while specific nitrogenase activity of nodules in Ar:O₂ was 50% of nodules supplied Air. Results indicated that nitrogen assimilation, whether from N₂ fixation or inorganic sources, had a localized effect on root development. Nodule development accounted for the major decrease in total photosynthate partitioning to non-N₂-fixing nodules. Soybean compensates for ineffective nodulation by controlling the flux of carbon to ineffective nodules and their associated roots.     

N(2)-Fixation by Freshly Isolated Nostoc from Coralloid Roots of the Cycad Macrozamia riedlei (Fisch. ex Gaud.) Gardn

Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation (¹⁵N₂ fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and ¹⁵N₂ fixation in light. The relationship between ethylene formation and N₂-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C₂H₄ per nanomole ¹⁵N₂. Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O₂ in light, while in darkness there was a broad optimum around 5 to 8% O₂. Inhibition of nitrogenase, in light, by pO₂ above 0.6% was irreversible. Measurements of light-dependent O₂ evolution and ¹⁴CO₂ fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O₂ levels above 6% O₂ and the inhibition was reversible. These cells showed rates of light-dependent O₂ evolution and ¹⁴CO₂ fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O₂, and a direct dependence on the cycad for substrates to support nitrogenase activity.     

Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO₂ production) and nitrogenase activity (H₂ production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N₂:O₂, thus under N₂-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N₂ deprivation that occur in acetylene reduction assays and during extended Ar:O₂ exposures in H₂ assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O₂ from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO₂/H₂. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO₂ produced per 2e^– transferred by nitrogenase to reduction of N₂. The results ranged from 2.6 to 3.4mol CO₂ produced per 2e^–. Carbon cost expressed as gC produced per gN reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N₂-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.     

Effect of supra-ambient oxygen on nitrogenase activity (c(2)h(2)) and root respiration of soybeans and isolated soybean bacteroids

Isolated soybean (Glycine max [L.] Merr. cv Wilkin) bacteroids have O₂-dependent nitrogenase activity which is strongly inhibited by supraoptimal O₂ concentrations. Oxygen-inhibited nitrogenase activity is recovered by addition of 10 millimolar sodium succinate or by lowering the O₂ concentration.

Brief treatment of roots of intact soybean plants with 1.0 atmosphere O₂ reduces nitrogenase activity (C₂H₂). There is a rapid partial recovery of activity within 2 to 3 hours, and a slower return to near normal levels by 36 hours. The drop and recovery of nitrogenase activity is accompanied by a parallel drop and increase in root respiration. There is a direct relationship between the change in respiration and the change in acetylene reduction following O₂ treatment. The O₂-mediated changes in nitrogenase activity and root respiration are not affected by the planting medium. The ratio of the change in respiration to the change in nitrogenase activity was the same in 13 soybean cultivars.