Acetylene Reduction Rates Research Articles

Response to Soil Temperature of Dinitrogen Fixation (Acetylene Reduction) Rates by Field‐Grown Soybeans1

AbstractTemperature has a major effect on the dinitrogen (N2) fixation rate of legume nodules. Experiments were performed under field conditions to assess the influence of soil temperature on acetylene reduction rates by soybean [Glycine max(L.) Merr.]. Data were collected in situ under low acetylene concentrations such that observations of a number of plants were made every 30 min throughout diurnal periods. Significant positive correlations existed between acetylene reduction rates and soil temperature at temperatures below 30 °C. A linear model and an exponential model fit the data equally well over the 10 to 12 °C ranges where data were obtained. Since it was determined that both the nodule gas conductance and maximum activity of the nodule changed in parallel, it was not possible to identify which of these terms was responding directly to the changes in soil temperature. Between 30 and 33 °C little change in acetylene reduction rates with soil temperature was observed. Above 34 °C declines in acetylene reduction rates were observed. The decline was usually to 70% of the values observed in the 30 to 33 °C soil temperature range. Little or no recovery was observed in acetylene reduction rates after this high‐temperature–induced decline, indicating that a more permanent alteration had occurred in the nodules.

Diurnal and Seasonal Variation in Dinitrogen Fixation (Acetylene Reduction) Rates by Field–Grown Soybeans1

AbstractNo data exist that represents in situ, continuous observations under field conditions of changes in dinitrogen fixation potential through diurnal cycles. Acetylene reduction responses of soybean [Glycine max(L.) Merr.] plants to changes during the diurnal cycle and during seasonal developmental changes were monitored using a continuous flow system with very low acetylene concentration. These data were used to assess the relative effects on acetylene reduction rates of environmental and plant ontogeny variables. During the diurnal cycle, acetylene reduction rates generally peaked late in the afternoon and then declined through the night. However, the night rates did not fall below half of the daily maximum. Within individual days, rates were more closely correlated with soil temperature than with photosynthetically active radiation. A consistent link was not observed between acetylene reduction rates and any particular stage of ontogeny. These results, and a reexamination of published reports, suggest that dinitrogen fixation rate in soybeans is not closely coupled to current photosynthetic rates under field conditions.

Biological and Cultural Characteristics of the Effective Frankia Strain HFPCcl3 (Actinomycetales) from Casuarina cunninghamiana (Casuarinaceae)

From homogenates prepared from surface-sterilized nodules of seedlings of Casuarina cunninghamiana grown aeroponically, a strain of Frankia designated HFPCc13 was isolated and has been grown in pure filamentous culture in a defined synthetic nutrient medium. Vesicle and sporangium formation can be induced by removal of combined nitrogen from the medium.Frankia strain HFPCc13 nodulates young seedlings of C. cunninghamiana and C. equisetifolia within three weeks of inoculation with an optimum root medium pH of 6–7 for nodulation and optimum temperature of 30–35 °C. The presence of combined nitrogen in the root medium inhibits nodulation with NH4+ more inhibitory than NO3−. Frankia HFPCc13 does not nodulate Allocasuarina species within the same family nor several other possible actinorhizal plants tested. Thus this strain is quite precise in its host specificity. The rate of acetylene reduction was greater in C. cunninghamiana than the closely related species C. equisetifolia. In neither of these host species were vesicles observed to occur within the infected root nodules which had been demonstrated to be actively fixing dinitrogen. Root nodules were shown to be active in acetylene reduction over a range of O2 concentration in the gaseous environment with an optimum at about 20 per cent O2, the ambient PO2 of the air. The mechanism(s) for oxygen protection of nitrogenase within the filamentous form of Frankia within these nodules remains to be explained.

Effect of O2 on vesicle formation, acetylene reduction, and O2-uptake kinetics in Frankia sp. HFPCcI3 isolated from Casuarina cunninghamiana.

The effect of the partial pressure of oxygen (PO2) on the formation of vesicles, which are thought to be the site of N2 fixation in Frankia, was studied in HFPCcI3, an effective isolate from Casuarina cunninghamiana. Unlike other actinorhizal root nodules, vesicles are not produced by the endophyte in Casuarina nodules. However, in culture under aerobic conditions, large, phase-bright vesicles are formed in HFPCcI3 within 20 h following removal of NH+4 from the culture medium and reach peak numbers within 72 to 96 h. In vivo acetylene reduction activity parallels vesicle formation. Optimum rates of acetylene reduction in short-term assays occurred at 20% O2 (0.2 atm (1 atm = 101.325 kPa] in the gas phase. O2 uptake (respiration) determined polarographically showed diffusion-limited kinetics and remained unsaturated by O2 until 300 microM O2. In contrast, respiration in NH+4-grown cells was saturated by O2 between 8 and 10 microM O2. These results indicate the presence of a diffusion barrier associated with the vesicles. Vesicle development was repressed in cells incubated in N-free media sparged with gas mixtures with PO2 between 0.001 and 0.003 atm. Nitrogenase was induced under these conditions, but acetylene reduction was extremely O2 sensitive. The kinetics of O2 uptake as a function of dissolved O2 concentration in avesicular cells were similar to those in NH+4-grown cells indicating the lack of a diffusion barrier. These results demonstrate that vesicle formation and the development of the O2 protection mechanisms of nitrogenase are regulated by ambient PO2 in HFPCcI3.

H2-dependent mixotrophic growth of N2-fixing Azotobacter vinelandii.

Azotobacter vinelandii can grow with a variety of organic carbon sources and fix N2 without the need for added H2. However, due to an active H2-oxidizing system, H2-dependent mixotrophic growth in an N-free medium was demonstrated when mannose was provided as the carbon source. There was no appreciable growth with either H2 or mannose alone. Both the growth rate and the cell yield were dependent on the concentrations of both substrates, H2 and mannose. Cultures growing mixotrophically with H2 and mannose consumed approximately 4.8 mmol of O2 and produced 4.6 mmol of CO2 per mmol of mannose consumed. In the absence of H2, less CO2 was produced, less O2 was consumed, and cell growth was negligible. The rate of acetylene reduction in mixotrophic cultures was comparable to the rate in cultures grown in N-free sucrose medium. The rate of [14C]mannose uptake of cultures with H2 was greater than with argon, whereas [14C]sucrose uptake was unaffected by the addition of H2; therefore, the role of H2 in mixotrophic metabolism may be to provide energy for mannose uptake. A. vinelandii is not an autotroph, as attempts to grow the organism chemoautotrophically with H2 or to detect ribulose bisphosphate carboxylase activity were unsuccessful.

Response to Drought Stress of Nitrogen Fixation (Acetylene Reduction) Rates by Field-Grown Soybeans

The effects of drought stress on soybean nodule conductance and the maximum rate of acetylene reduction were studied with in situ experiments performed during two seasons and under differing field conditions. In both years drought resulted in decreased nodule conductances which could be detected as early as three days after water was withheld. The maximum rate of acetylene reduction was also decreased by drought and was highly correlated with nodule conductance (r = 0.95). Since nodule conductance is equal to the nodule surface area times the permeability, the relationship of these variables to both whole-plant and unit-nodule nitrogenase activity was explored. Drought stress resulted in a decrease in nodule gas permeability followed by decreases in nodule surface area when drought was prolonged. Under all conditions studied acetylene reduction on a unit-nodule surface area basis was highly correlated with nodule gas permeability (r = 0.92). A short-term oxygen enrichment study demonstrated nodule gas permeability may limit oxygen flux into both drought-stressed and well-watered nodules of these field-grown soybeans.

Aspects of nitrogen fixation in Lough Neagh. I. Acetylene reduction and the frequency of Aphanizomenon flos‐aquae heterocysts

SUMMARY. An investigation was made of the relationships between rates of nitrogen fixation (acetylene reduction), Aphanizomenon flosaquae heterocysts, NO3‐N concentration and other environmental factors which occurred in Lough Neagh between 1969 and 1972. The maxima of cells differentiated as heterocysts (approximately 5% (1970), and approximately 7% (1971 and 1972) occurred when NO3‐N concentration was very low or undetectable. Acetylene reduction was detectable between 16 May and 8 September 1972 but was not measured in the other years. Rates were greatest in the euphotic zone and it appeared that the nitrogenase activity was closely linked to a light dependent system. Areal rates of acetylene reduction were highly correlated with Aphanizomenon heterocyst concentration (r=0.92). There was some indication that a release of phosphorus from the sediments might have stimulated Aphanizomenon nitrogen fixation on one occasion at the end of a calm period. Results show that the most likely conditions for the development of large crops of heterocystous species in Lough Neagh include a scarcity of NO3‐N and a plentiful supply of phosphorus.

H2, N2, and O2 metabolism by isolated heterocysts from Anabaena sp. strain CA.

Metabolically active heterocysts isolated from wild-type Anabaena sp. strain CA showed high rates of light-dependent acetylene reduction and hydrogen evolution. These rates were similar to those previously reported in heterocysts isolated from the mutant Anabaena sp. strain CA-V possessing fragile vegetative cell walls. Hydrogen production was observed with isolated heterocysts. The ratio of C2H4 to H2 produced ranged from 0.9 to 1.2, and H2 production exhibited unique biphasic kinetics consisting of a 1 to 2-min burst of hydrogen evolution followed by a lower, steady-state rate of hydrogen production. This burst was found to be dependent upon the length of the dark period immediately preceding illumination and may be related to dark-to-light ATP transients. The presence of 100 nM NiCl2 in the growth medium exerted an effect on both acetylene reduction and hydrogen evolution in the isolated heterocysts from strain CA. H2-stimulated acetylene reduction was increased from 2.0 to 3.2 mumol of C2H4 per mg (dry weight) per h, and net hydrogen production was abolished. A phenotypic Hup- mutant (N9AR) of Anabaena sp. strain CA was isolated which did not respond to nickel. In isolated heterocysts from N9AR, ethylene production rates were the same under both 10% C2H2-90% Ar and 10% C2H2-90% H2 with or without added nickel, and net hydrogen evolution was not affected by the presence of 100 nM Ni2+. Isolated heterocysts from strain CA were shown to have a persistent oxygen uptake of 0.7 mumol of O2 per mg (dry weight) per h, 35% of the rate of whole filaments, at air saturating O2 levels, indicating that O2 impermeability is not a requirement for active heterocysts.

Effect of Nitrogen Supply on the Grass and Clover Components of Simulated Mixed Swards Grown under Favourable Environmental Conditions II. Nitrogen Fixation and Nitrate Uptake

Simulated mixed swards of Perennial Ryegrass (Lolium perenne L.) cv. S23 and White clover (Trifolium repens L.) cv. S100 were grown from seed under a constant 20 °C day/15 °C night temperature regime and harvested at intervals over an d88 d growht period. The swards received a nutrient solution daily, which was either High (220 mg l−1) or Low (10 mg l−1) in nitrate N. The nitrate was labelled with the 15N isotope. An acetylene reduction assay was carried out on each sward just prior to harvest. Rates of acetylene reduction agreed qualitatively with the l5N analyses but absolute values did not match (assuming a 4:1 C2H4:N2 ratio) and errors in the acetylene assay are discussed. In the Low-N swards clover relied almost entirely on symbiotically fixed N2, fixing more than ten times as much as the High-N clover plants. In the Low-N treatment the grass was N-deficient despite obtaining much more nitrate per unit root dry weight than clover. In the High-N swards, however, clover took up more nitrate per unit root weight than grass. The High-N clover plants also fixed some N2 and maintained a higher total-N content than grass throughout the period. There was no evidence of transfer of symbiotically fixed N from the clover to the grass in either treatment.

Nitrogen fixation in the high arctic tundra at Sarcpa Lake, Northwest Territories

The acetylene reduction assay was used to examine biological nitrogen fixation in the high arctic tundra at Sarcpa Lake, Northwest Territories (68°32′ N, 83°19′ W). The highest rates of acetylene reduction (9.37 ± 3.19 μmol C2H4 m−2 h−1) were in habitats that had a high density of the legumes Oxytropis maydelliana, O. arctobia, and Astragalus alpinus. Nitrogen fixation in the wet soils along the shore of a small lake was similar (8.87 ± 4.35 μmol C2H4 m−2 h−1) because of the blue-green alga Nostoc, which associates with mosses. Free-living blue-green algae and lichens made insignificant contributions to the total nitrogen fixation budget because they were uncommon and fixed nitrogen at a slower rate. Nitrogen-fixing lichens in the area included Stereocaulon arenarium and S. rivulorum. It is concluded that legumes have a significant input to the biological nitrogen fixation budget at Sarcpa Lake.

Nitrogen Fixation Associated with the New Zealand Mangrove ( Avicennia marina (Forsk.) Vierh. var. resinifera (Forst. f.) Bakh.)

Nitrogenase activity in mangrove forests at two locations in the North Island, New Zealand, was measured by acetylene reduction and N(2) uptake. Nitrogenase activity (C(2)H(2) reduction) in surface sediments 0 to 10 mm deep was highly correlated (r = 0.91, n = 17) with the dry weight of decomposing particulate organic matter in the sediment and was independent of light. The activity was not correlated with the dry weight of roots in the top 10 mm of sediment (r = -0.01, n = 13). Seasonal and sample variation in acetylene reduction rates ranged from 0.4 to 50.0 mumol of C(2)H(4) m h under air, and acetylene reduction was depressed in anaerobic atmospheres. Nitrogen fixation rates of decomposing leaves from the surface measured by N(2) uptake ranged from 5.1 to 7.8 nmol of N(2) g (dry weight) h, and the mean molar ratio of acetylene reduced to nitrogen fixed was 4.5:1. Anaerobic conditions depressed the nitrogenase activity in decomposing leaves, which was independent of light. Nitrogenase activity was also found to be associated with pneumatophores. This activity was light dependent and was probably attributable to one or more species of Calothrix present as an epiphyte. Rates of activity were generally between 100 and 500 nmol of C(2)H(4) pneumatophore h in summer, but values up to 1,500 nmol of C(2)H(4) pneumatophore h were obtained.

The Effect of Nickel on Hydrogen Metabolism and Nitrogen Fixation in the Cyanobacterium Anabaena cylindrica

A comparative study was made of the growth and nitrogen fixation of nickel-depleted and nickel-supplemented cultures of the cyanobacterium Anabaena cylindrica. Four sets of growth conditions were used, involving both dark/light and continuous light regimes, anaerobic and aerobic conditions, light limitation and supplementation of the gas phase with hydrogen. In each case nickel-containing cells had an active hydrogen uptake capacity whereas nickel-depleted cells did not. These differences in hydrogenase activities did not correlate with differences in acetylene reduction and growth rates, or fixed nitrogen, phycocyanin or chlorophyll contents. It is concluded that under the growth conditions used the capacity of cells to consume hydrogen gas confers no advantage to the organisms in terms of their growth rates and nitrogen fixation.

Relationship Between Acetylene Reduction and Dinitrogen Fixation in Pisum sativum L. and Phaseolus vulgaris L.

Summary Quantitative relationships between the rate of acetylene reduction (nitrogenase activity) and the accumulation of symbiotically fixed dinitrogen were studied. Some methodical aspects of seasonal, short term and diurnal fluctuations of nitrogenase activity, together with dihydrogen evolution data, are reported and discussed. The results reveal that quantitative interpretations of C2H2 reduction data do not mean overestimation of symbiotic dinitrogen fixation.

Structure of Nodules Formed by Rhizobium Strain ANU289 in the Nonlegume Parasponia and the Legume Siratro (Macroptilium atropurpureum)

A study of root nodules produced by Rhizobium strain ANU289 in the legume siratro (Macroptilium atropurpureum) and the nonlegume Parasponia rigida showed that the legume nodule has a "typical" tropical legume nodule morphology: spherical determinate nodules with a peripheral vascular system. The invaded zone contains both infected hypertrophied cells and small uninfected starch-containing interstitial cells. All infected cells are at a comparable developmental stage. The bacteroids are packaged in the peribacteroid membrane that is of plant origin. Typically three to four bacteroids are found per enveloping membrane. The nonlegume nodule is a modified lateral root with multiple regions of apical meristematic activity; the nodule is indeterminate and has a centralized vascular system. Developmentally, the infected cells vary; the mature, fully infected cells are most distant from the meristem. The bacteroids are not released from the membrane-bound infection thread, which appears to develop into a lightly staining, thin-walled thread (the "fixation" thread) that contains numerous, loosely packed bacteroids, often with significant deposits of polybetahydroxybutyrate. The morphology of the bacteroids is similar in both nodule types, which are capable of high rates of acetylene reduction.

Consequences of Sporangial Development for Nodule Function in Root Nodules of Comptonia peregrina and Myrica gale

Frankia sp., the actinomycetous endophyte in nitrogen-fixing actinorhizal nodules, may differentiate two forms from its hyphae: vesicles and sporangia. In root nodules of Comptonia peregrina (L.) Coult. and Myrica gale L., sporangia may be either absent or present. Nitrogenase activity and symbiotic efficiency were contrasted in spore(+) and spore(-) nodules of these two host genera. Seedlings of C. peregrina nodulated with the spore(+) inoculum showed only 60% of the nitrogenase activity and 50% of the net size of their spore(-) counterparts after 12 weeks of culture. Measurements of acetylene reduction (i.e., nitrogenase activity) were coordinated with samplings of nodules for structural studies. Significant differences in acetylene reduction rates were discernible between spore(+) and spore(-) nodules commencing 4 weeks after nodulation, concomitant with the maturation of sporangia in the nodule. Spore(+) nodules ultimately reached less than half of the rate of nitrogenase activity of spore(-) nodules. Both types of nodules evolved only small amounts of molecular hydrogen, suggesting that both were equally efficient in recycling electrons lost to the reduction of hydrogen ions by nitrogenase. Respiratory cost of nitrogen fixation, expressed as the quotient of micromole CO(2) to micromole ethylene evolved by excised nodules, was significantly greater in spore(+) than in spore(-) nodules. M. gale spore(-) nodules showed variable effectivity, though all had low CO(2) to ethylene evolution ratios. M. gale spore(+) nodules resembled C. peregrina spore(+), with low effectivity and high respiratory cost for nitrogen fixation.

Oxygen protection of nitrogenase in Frankia sp. HFPArI3.

O2 protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O2 uptake and acetylene reduction rates in response to substrate concentration. O2 of NH4+-grown cells showed an apparent KmO2 of approximately 1 microM O2. In N2-fixing cultures a second Km O2 of about 215 microM O2 was observed. Thus, respiration remained unsaturated by O2 at air-saturation levels. In vivo, the apparent Km for acetylene was more than 10-fold greater than reported in vitro values. These data were interpreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46 degrees 54' S, 37 degrees 45' E) increased from -5 degrees C to a maximum at 25 to 27 degrees C. Q(10) values between 0 and 25 degrees C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35 degrees C than at 0 degrees C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 mumol m s, saturation occurring at ca. 1,000 mumol m s. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.

Host Plant Nodule Enzymes Associated with Selection for Increased N2 Fixation in Alfalfa1

In legumes symbiotically fixed N2 is assimilated into C skeletons to form amino acids as a prerequisite for enhancing plant growth. No attempts have been made to determine which nodule enzymes of ammonia and C assimilation are related to N2 fixation in alfalfa (Medicago sativa L.). The objective of this study was to assess if nodule enzymes of ammonia and C assimilation were associated with acetylene reduction acitivity in alfalfa genotypes previously selected for various traits associated with N2 fixation. Nodules from eight subpopulations derived by bi‐directional selection within each of two alfalfa germplasm sources, an intercross between the two germplasm sources and three alfalfa cultivars were used to measure nodule soluble protein, and in vitro specific activities of the plant enzymes glutamine synthetase (GS), glutamate synthase (GOGAT) glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxylase (PEPC). Acetylene reduction rate was measured as an indicator of nitrogenase activity. Significant genotypic differences were observed among subpopulations for all variables tested. The GOGAT and PEPC activities were correlated with acetylene reduction activity in one germplasm source but not the other. Phenotypic correlation coefficients among variables showed that GOGAT and PEPC activities were frequently coupled. Germplasm differences in nodule enzyme activity suggest that nodule carbon and N assimilation may be more closely associated with N2 fixation potential in one germplasm than the other. The GOGAT and PEPC activity may be useful traits to include in breeding programs to enhance N2 fixation in alfalfa.

Growth and root nodule nitrogenase activity of Pisum sativum as influenced by transpiration

The influence of shoot transpiration on the rates of growth and nitrogen fixation was investigated in Pisum sativum L. cv. Rondo. In short term experiments, rates of transpiration and acetylene reduction of intact plants were measured simultaneously, using air‐tight perspex vessels enclosing the basal part of the nodulated root. In long term experiments, accumulation of dry matter and reduced nitrogen in the plant were determined as well. Transpiration rate changed diurnally and was varied by manipulating the vapour saturation deficit or the flow rate of the air in the growth cabinet. The rate of acetylene reduction declined after subjecting intact plants to high transpiration rates. This decline was accompanied by a desiccation of the root nodules. Dry matter and reduced nitrogen accumulation were not affected by transpiration rate. At low transpiration rate reduced nitrogen content of the root nodules was higher than at high transpiration rate. However, in these nodules the rate of acetylene reduction was not significantly affected. It is concluded that the nitrogenase activity of pea root nodules is insensitive to changes in the flow rate and the organic N concentration of the xylem sap within a wide range of transpiration conditions under the applied growth conditions.

Growth and acetylene reduction of black alder seedlings in response to water stress

Black alder, Alnusglutinosa (L.) Gaertn., seedlings were grown and kept well watered for 10 weeks, and then subjected to moisture stress conditioning for 5 subsequent weeks, where one-half of the seedlings were watered only when visibly wilted. The remaining seedlings (controls) were kept well watered. Moisture stress conditioning greatly reduced shoot, root, nodule, and total plant dry weight. The root–shoot ratio (grams/grams) of seedlings was significantly increased from 0.28 in the control seedlings to 0.33 in the water-stressed treatment. Acetylene reduction rates decreased only slightly in the range of water potentials between −0.50 and −1.29 MPa, then dropped rapidly below water potentials of −1.30 MPa. Moisture stress conditioning had no significant influence on this response. Although not significantly different, leaf and nodule osmotic potentials were consistently lower in the water-stressed plants.