Field technique using the acetylene reduction method to assay nitrogenase activity and its association with the rice rhizosphere

Summary A plastic bag, used as a field assay chamber, assisted in the investigation of the acetylene reduction method as a technique to measure nitrogen-fixing activity in rice paddy fields. A study of the change in volume of the plastic bag and of the loss of acetylene and ethylene from the bag provided evidence that this plastic bag method was feasible in field assays. The field assay of rice at the grain ripening stage showed that the nitrogen-fixing activity increased linearly after a lag phase of 0 to 3 hours during a time-course experiment.Detachment of the aerial part of the rice plant from its root remaining in the field did not affect the nitrogen-fixing activity of the root.The markedly higher nitrogen-fixing activity in the planted areas of the field compared with the nonplanted areas between the plant rows indicated that the nitrogenase activity in the field is associated with the roots of the rice plant. re]19751204     

Measurement of nitrogenase activity in legume root nodules: In defense of the acetylene reduction assay

The closed acetylene reduction assay has been used as a measure of nitrogenase activity and an indicator of N₂ fixation in Rhizobium/legume symbioses for 25 years. However, starting 10 years ago this assay has come under harsh criticism as being inaccurate. Currently, confusion exists regarding the conditions under which the acetylene reduction assay can be used accurately, or whether it can be used at all as a measure of nitrogenase activity. This article reviews the circumstance that has lead to this confusion. The author argues that under the proper assay conditions and with the appropriate checks, the closed acetylene reduction assay is still a valuable tool in assessing relative differences in nitrogenase activity in Rhizobium/legume symbioses.     

Non-biological production of ethylene in the acetylene reduction assay for nitrogenase

Summary Ethylene is formed as a result of contact between trichloroacetic acid and the rubber serum cap liners commonly used in the acetylene reduction assay for nitrogenase activity. The amount of ethylene produced varies from one rubber liner to another, but the rate is approximately linear over an eight-day experimental period. Immediate withdrawal of a sample of the gas phase from the incubation bottle at the end of the experiment, and storage in another vessel is recommended as an alternative to the addition of any substance to terminate experiments.     

The acetylene reduction technique as an assay for nitrogenase activity in the methane oxidizing bacterium Methylococcus capsulatus strain bath

The use of acetylene as a convenient assay substrate for nitrogenase in methane oxidising bacteria is complicated by the observation that it is a potent inhibitor of the methane monooxygenase enzyme in both whole cells and cell-free extracts. If the cells were provided with alternative oxidisable carbon substrates other than methane then nitrogen fixing cells would reduce acetylene to ethylene. Hydrogen gas also served as an oxidisable substrate in the assay. Nitrous oxide, which is reduced by nitrogenase to N₂ and H₂O, was not an inhibitor of methane monooxygenase function and could be used as a convenient assay substrate for nitrogenase. Reduction of both substrates by whole cells showed similar response to oxygen in the assay system and in this respect Methylococcus resembles other free living nitrogen fixing aerobes.     

The effect of conditions of low oxygen tension on the assay of nitrogenase in moulds and yeasts using the acetylene reduction technique

Summary 15 strains of Pullularia, Rhodotorula, Bullera and Torulopsis were tested for acetylene reduction under anaerobic and low pO₂ (0.05) conditions. No production of ethylene was observed and it is concluded that nitrogenase activity is absent.     

Laboratory acetylene reduction assay for relative measurement of N2-fixing activities associated with field-grown wetland rice plants

Summary A short-term laboratory acetylene reduction assay using cut plant-soil samples incubated in the dark was developed for measuring relative N₂-fixing activities associated with field-grown rice plants. The assay sample consists of rhizosphere soil, root, and cut stem and leaf sheath. The cut plant-soil assay is relatively simple, rapid, and convenient; it reduces, if not eliminates, the problems encountered in whole-plant (field, pot, and water culture) and excised roots assays. Varietal differences in N₂-fixing activity were detected with the new assay technique.     

Heterotrophic nitrogen fixation (acetylene reduction) associated to flooded rice: A modified measurement technique in the field

A modifiedin situ technique for measuring heterotrophic nitrogen fixing (acetylene reducing) activity associated to rice is proposed. Ethylene evolution rates measured in opaque cylinders covering the stems of rice plants which have been cut 10 cm over the water level were found independent of the diurnal cycle. Cutting of the leaves resulted in decreased variation between plants and suppression of the acceleration of ethylene evolution rate after 12 h incubation as compared to intact plants. In both systems ethylene evolved was swept by a current of methane and the molar ratio between methane and ethylene was stabilized after 12 h. Methane evolution rates remained stable during 12 h and more than 24 h in whole plants and cut plants respectively. It is suggested that alteration in the active gas transport system after 12 h incubation under 10% acetylene may lead to erroneous evaluation of the actual ethylene production in the root's environment. The average values of ethylene evolution rates by cut plants between 12 and 24 h of incubation may be used for comparative studies of nitrogen fixing activity associated to flooded rice.     

The acetylene reduction assay inactivates root nodule uptake hydrogenase in some actinorhizal plants

Actinorhizal nodules do not usually evolve H₂ due to the action of an uptake hydrogenase. We have found that nodules of several Frankia symbioses evolved large amounts of H₂ gas when returned to air following exposure to 10 kPa C₂HT₂ during an acetylene reduction assay. Increased H₂ evolution in air persisted for several days when intact root systems of Alnus incana (L.) Moench (inoculated with Frankia UGL 011101) were treated with 10 kPa C.H₂ for 1 h. Full recovery of uptake hydrogenase activity required 4 to 8 days. Studies with crude homogenates of nodules of the same plants showed that hydrogenase (measured amperometrically with phenazine metho-sulfate as electron acceptor) was directly affected, since activity in treated nodules was only 10% of that in untreated nodules. A survey of actinorhizal symbioses revealed variation in the effect of an acetylene reduction assay on hydrogen metabolism. Nodules of three species, including Alnus rubra Bong, inoculated with Frankia HFPArD. showed complete inactivation of hydrogenase. H₂ evolution in air was 25% of the C₂H₂ reduction rate and H, evolution in Ar/O₂ was equal to the QH₂ reduction rate. Two symbioses, Ceanothus americanus L. (soil inoculant) and Batista glomerata Baill. (soil inoculant) showed no change following an acetylene reduction assay. A third group of symbioses showed an intermediate response.     

Simultaneous measurement of acetylene reduction and respiratory gas exchange of attached root nodules

A method was developed for the simultaneous measurement of acetylene reduction, carbon dioxide evolution and oxygen uptake by individual root nodules of intact nitrogen-fixing plants (Alnus rubra Bong.). The nodules were enclosed in a temperature-controlled leak-tight cuvette. Assay gas mixtures were passed through the cuvette at a constant, known flow rate and gas exchange was measured by the difference between inlet and outlet gas compositions. Gas concentrations were assayed by a combination of an automated gas chromatograph and a programmable electronic integrator. Carbon dioxide and ethylene evolution were determined with a coefficient of variation which was less than 2%, whereas the coefficient of variation for oxygen uptake measurements was less than 5%. Nodules subjected to repeated removal from and reinsertion into the cuvette and to long exposures of 10% v/v acetylene showed no irreversible decline in respiration or acetylene reduction. This system offers long-term stability and freedom from disturbance artifacts plus the ability to monitor continuously, rapidly and specifically the changes in root nodule activity caused by environmental perturbation.     

An acid precipitation technique: A strip assay for the large-scale DNA polymerase activity screening

A new format of a very rapid, low-cost and high-productive analysis based on the acid precipitation of radiolabeled DNA was developed. By contrast to the conventional processing of a large number of GF/C discs, the method employs one GF/C strip containing samples on individual teeth. The strip assay was validated by comparison with the glass fiber disk technique; the efficiency was demonstrated by screening E. coli superproducers and fractions obtained at the steps of Bst DNA polymerase, Large Fragment purification by the protocol we developed. The principle proposed allows simultaneous assaying many samples for the activity of different polymerases.     

A non-destructive field assay for soybean nitrogen fixation by acetylene reduction

Summary A system for employing open-ended root chambers to measurein situ acetylene reduction rates under field conditions is described. Gas mixtures containing about 2 mbar acetylene were continuously flowed through the chambers providing a continuous record of acetylene reduction. These chambers have been used to measure acetylene reduction rates of soybeans during three growing seasons. The system has proved to be reliable with a high degree of precision. The large amount of plant-to-plant variability observed in N₂ fixation research has been confirmed by the data collected with this system. However, such variability in physiological studies can be reduced by using a non-destructive system to compare the response of an individual plant with its rates before treatment.     

Emerson enhancement of carbon fixation but not of acetylene reduction (nitrogenase activity) in Anabaena cylindrica

Summary Enhancement of carbon fixation was demonstrated in the bluegreen alga, Anabaena cylindrica, grown in either aerobic or microaerobic conditions. Under identical conditions no enhancement of acetylene reduction was observed. Light absorbed by photosystem I supported relatively more acetylene reduction than carbon fixation. No competition between the two processes was observed under light-limiting conditions. The findings suggest that carbon fixation and acetylene reduction may depend on different pools of reductant and ATP. When aerobically grown cells were placed in the dark or at limiting light intensities, acetylene reduction was higher in air than under argon. In contrast, carbon fixation was lower in air than in argon.     

Improved method for preparing anaerobic bacteroid suspensions of Rhizobium leguminosarum for the acetylene reduction assay.

A method using ethylenediaminetetraacetate (EDTA) and toluene-treated pea bacteroid suspensions for the acetylene reduction assay is described. The high level of acetylene reduction by these bacteroids is comparable to that of intact plants. Reproducibility of the EDTA-toluene treatment is, on the average, within 5%. Preliminary experiments with soybeans indicate that the EDTA-toluene method might be applicable to other legumes as well.     

Factors affecting vesicle formation and acetylene reduction (nitrogenase activity) in Frankia sp. CpI1

Vesicle formation and acetylene reduction (nitrogenase activity) were observed when washed hyphae from cultures of Frankia sp. CpI1 were transferred to a nitrogen-free medium containing ethylenediaminetetraacetic acid and succinate. Succinate could be replaced by malate or fumarate, but not other carbon sources. Maximum acetylene reduction and vesicle numbers were observed at a pH of 6.0-6.5, at 25-30 degrees Centigrade, and at atmos pheric Po2 or somewhat less (5-20 kPa). Addition of 1 mM NH4Cl almost completely inhibited vesicle formation and acetylene-reducing activity, but did not immediately inhibit such reducing activity by cultures with preexisting vesicles. Acetylene-reducing activity was never observed in the absence of vesicle formation.     

Klebsiella pneumoniae nitrogenase. Mechanism of acetylene reduction and its inhibition by carbon monoxide.

Spontaneous decomposition of 5-phosphoribosyl 1-pyrophosphate at pH 5.5 was established to occur as follows: 5-Phosphoribosyl 1-pyrophosphate----5-phosphoribosyl 1,2-(cyclic)phosphate----ribose 1-phosphate----ribose Enzymic degradation of 5-phosphoribosyl 1-pyrophosphate by alkaline phosphatase from calf intestine and by acid phosphatases from potato and Aspergillus niger was found to proceed according to this pathway within the pH range 2.5-7.4 with accumulation of ribose 1-phosphate. In the case of alkaline phosphatase, Mg2+ ions inhibit the pyrophosphorolysis of 5-phosphoribosyl 1-pyrophosphate and stimulate the hydrolysis of ribose 1-phosphate.     

Effects of concentrating phytoplankton on the acetylene-reduction assay for nitrogenase activity

SUMMARY.

• 1 The consequences of concentrating freshwater phytoplankton communities prior to measurement of nitrogenase activity using the acetylene-reduction assay were investigated.
• 2 Retention of heterocystous blue-green algae by 10 and 45 öm plankton nets was usually much less than 100% and the percent retention differed among blue-green algal species and varied for the same species both on different sampling dates and in different lakes. Retention of heterocyst numbers and helerocyst volume during concentration differed from that of total cell volume of heterocystous blue-green algae.
• 3 Effects of concentration on specific nitrogcnase activity varied among the lakes studied. In Lake Bysjön activity decreased on all sampling occasions, in Lake Trummen there was no apparent effect, and in Lake Vaxjosjon both results were found.
• 4 Effects on nitrogenase activity of mechanical disturbance during concentration and of increased pH. reduced carbon availability. light inhibition and subsaturation. photorespiration and oxygen supersaturation in concentrated samples are discussed. Interactions between these factors are suggested to explain the varying responses in the three lakes.
• 5 It is concluded that phytoplankton should not be concentrated in the acetylene-reduction assay for nitrogenase activity, since both volumetric and cell concentration factors are inadequate and effects of concentration on specific nitrogenase activity are not predictable.

     

Nitrogenase activity in cyanobacteria measured by the acetylene reduction assay: a comparison between batch incubation and on-line monitoring

A new on-line method for measuring acetylene reduction is described. It consists of a gas-flow cell connected to an electronic gas-mixing system and an automatic sample loop in the gas chromatograph. Alternatively, ethylene can be determined by using laser-based trace gas detection. The laser-based trace gas detection technique achieves a detection limit that is three orders of magnitude better than gas chromatography. We have applied the on-line method to the measurement of nitrogen fixation in a culture of the heterocystous cyanobacterium Nodularia spumigena and compared it with conventional batch-type incubations. Incubation of N. spumigena in the gas-flow cell resulted in very short response times with a steady-state flux of ethylene obtained within 2 min. Nitrogenase was shown to respond immediately to changes in light and oxygen. Monitoring of nitrogenase activity could be continued for several hours without having a negative impact on nitrogen fixation rates in N. spumigena . This was not the case in batch incubations, in which changes in nitrogenase activities were recorded during incubations, probably as a result of varying oxygen concentrations. It was therefore concluded that the on-line method is superior to batch incubations when rates of nitrogenase activity are to be measured. The method is suitable for natural samples (water or sediment).     

An intact plant assay for estimating nitrogenase activity (C2H2 reduction) of sorghum and millet plants grown in pots

Summary A non destructive intact-plant assay for estimating nitrogenase activity (C₂H₂ reduction) of pot-grown sorghum and millet plants is described. Plants with intact shoots sustained more activity than plants whose tops were removed prior to the assay. With this technique individual plants can be assayed several times during their life cycle. The C₂H₂ reduction was linear up to 16h incubation in this assay procedure. More rapid diffusion of C₂H₂ was achieved by injection through a Suba seal in the bottom of the pot. The equlibration of injected C₂H₂ in the gas phase of the pots filled with sand and sand:FYM media was completed within 1 h. Significantly higher nitrogenase activity and better growth of sorghum and millet plants occurred when plants were grown in a mixture of sand and farmyard manure (FYM) than when plants were grown in vermiculite, soil, or sand + soil medium. Nitrogenase activity and plant growth were greater in a mixture of sand with 2 and 3% FYM than with 0.5 and 1% FYM. Activity was higher when the plants were incubated at 33°C and 40°C than at 27°C. Activity also increased with increasing soil moisture. There were significant differences amongst 15 sorghum cultivars screened for associated nitrogenase activity. This new technique has good prospects for screening cultivars of millet, sorghum and other grain crops for their nitrogen-fixing ability.Submitted as Journal article No. 358 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).