Nitrogen Fixation by Subterranean Clover at Varying Stages of Nodule Dehydration: II. EFFICIENCY OF NITROGENASE FUNCTIONING

Changes in nitrogenase activity (C₂H₂ reduction and H₂ production),nodulated root respiration and the efficiency of nitrogenasefunctioning were measured in response to progressive dehydrationof nodules on intact well-watered plants of subterranean clover(Trifolium subterraneum L.) cv. Seaton Park. The nodulated rootsof vegetative plants grown to the 14-leaf stage were incubatedin a gas exchange system through which a continuous dry airstreamwas passed over an 8 d period. The root tips were immersed inan N-free nutrient solution during this time so that water andion uptake was unimpeded. The decline in nodulated root respirationresulting from nodule drying was associated with a continualreduction in respiration coupled to nitrogenase activity. Asnodule water potential (_nod) decreased, the proportion of totalnodulated root respiration which was nitrogenase-linked declinedfrom 50% (day 1) to 33% (day 8). This was accompanied by a 79%reduction in specific nitrogenase activity (from 3.79 to 0.81umol C₂H₄ g^–1 nodule dry weight min^–1). Nodule dehydrationalso induced a decline in hydrogen (H₂) production in air. Therelative decline in hydrogen production exceeded that of acetylenereduction activity and this resulted in an increase in the relativeefficiency of nitrogenase functioning. However, the carbon costof nitrogenase activity progressively increased above 2.0 molCO₂ respired per mol C₂H₄ reduced as rood decreased below –0.4to –0.5 MPa. Consecutive measurements of the rates ofhydrogen evolution, ¹⁵N₂ fixation and acetylene reduction activityon intact unstressed plants resulted in a C₂H₄/N₂ conversionfactor of 4.08 and an electron balance of 1.08. These resultsindicated that the pre-decline rate of acetylene reduction activitymeasured in a flow-through system provided a valid measure ofthe total electron flux through nitrogenase. Key words: Subterranean clover, dehydration, efficiency, nitrogenase activity     

Defoliation in White Clover: Nodule Metabolism, Nodule Growth and Maintenance, and Nitrogenase Functioning During Growth and Regrowth

In two experiments, the functioning and metabolism of nodulesof white clover, following a defoliation which removed abouthalf the shoot tissue, were compared with those of undefoliatedplants. In one experiment, the specific respiration rates of nodulesfrom undefoliated plants varied between 1160 and 1830 µmolCO₂ g^–1h^–1, of which nodule ‘growth and maintenance’accounted for 22 ± 2 per cent, or 27 ± 3.6 percent, according to method of calculation. Defoliation reducedspecific nodule respiration and nodule ‘growth and maintenance’respiration by 60–70 per cent, and rate of N₂ fixationby a similar proportion. The original rate of nodule metabolismwas re-established after about 5 d of regrowth; during regrowthnodule respiration was quantitatively related to rate of N₂,fixation: 9.1 µmol CO₂ µmol^–1N₂. With the possible exception of nodules examined 24 h after defoliation,the efficiency of energy utilization in nitrogenase functioningin both experiments was the same in defoliated and undefoliatedplants: 2.0±0.1 µmol CO₂ µmol^–1 C₂H₄;similarly, there was no change in the efficiency of nitrogenasefunctioning as rate of N₂ fixation increased with plant growthfrom 1 to 22 µmol N₂ per plant h^–1. Exposure of nodulated white clover root systems to a 10 percent acetylene gas mixture resulted in a sharp peak in rateof ethylene production after 1.5–2.5 min; subsequently,rate of ethylene production declined rapidly before stabilisingafter 0.5–1 h at a rate about 50 per cent of that initiallyobserved. Regression of ‘peak’ rate of ethyleneproduction on rate of N₂ fixation indicated a value of 2.9 µmolC₂H₄ µmol^–1 N₂, for rates of N₂ fixation between1 and 22 µmol N₂ per plant h^–1. The relationshipsbetween nitrogenase respiration, acetylene reduction rates andN₂ fixation rates are discussed. Trifolium repens, white clover, defoliation, nodule respiration, N₂, fixation, nitrogenase     

Comparisons of the Respiratory Effluxes of Nodules and Roots in Six Temperate Legumes

The specific respiration rates of nodulated root systems, ofnodules and of roots were determined during active nitrogenfixation in soya bean, navy bean, pea, lucerne, red clover andwhite clover, by measurements on whole plants before and afterthe removal of nodule populations. Similar measurements weremade on comparable populations of the six legumes, lacking nodulesbut receiving abundant nitrate-nitrogen, to determine the specificrespiration of their roots. All plants were grown in a controlled-environmentclimate which fostered rapid growth. The specific respiration rates of nodulated root systems ofthe three grain and three forage legumes during a 7–14-dayperiod of vegetative growth varied between 10 and 17 mg CO₂g^–1 (dry weight) h^–1. This mean value consistedof two components: a specific root respiration rate of 6–9mg CO₂ g^–1 h^–1 and a specific nodule respirationrate of 22–46 mg CO₂ g^–1 h^–1. Nodule respirationaccounted for 42–70 per cent of nodulated root respiration;nodule weight accounted for 12–40 per cent of nodulatedroot weight. The specific respiration rates of roots lackingnodules and utilizing nitrate nitrogen were generally 20–30per cent greater than the equivalent rates of roots from nodulatedplants. The measured respiratory effluxes are discussed in thecontext of nitrogen nitrogen fixation, nitrate assimilation. Glycine max, Phaseolus vulgaris, Pisum sativum, Medicago sativa, Trifolium pratense, Trifolium repens, soya bean, navy bean, pea, lucerne, red clover, white clover, nodule respiration, root respiration, fixation, nitrate assimilation     

Nitrogen Fixation by Subterranean Clover at Varying Stages of Nodule Dehydration: I. CARBOHYDRATE STATUS AND SHORT-TERM RECOVERY OF NODULATED ROOT RESPIRATION

The nodule water potential (_nod) of subterranean clover (Trifoliumsubterraneum L.) cv. Seaton Park incubated in a flow-throughgas-exchange system was induced to decline independently ofleaf water potential (₁) by passing a continuous dry airstreamover the nodulated roots of intact well-watered plants. Reducedtranspiration by plants whose nodules had become dehydratedwas hypothesized to be related to the decline in nitrogen fixation.Whole-plant and nodule soluble carbohydrates increased as _noddeclined. Throughout an 8 d period of continual nodule dehydration,the gaseous diffusion resistance of nodules increased and theoptimum pO₂ for nitrogenase activity declined from 52 to 28kPa. Following rehydration of the nodulated roots between days4 and 5 and between days 7 and 8, nodulated root respirationincreased to or above pre-stress levels whereas nitrogenaseactivity did not recover. Re-establishment of initial ratesof nodulated root respiration was due to the stimulation ofgrowth and maintenance respiration, not to the respiration coupledto nitrogenase activity. Although no recovery of nitrogenaseactivity occurred, the elapsed time from the introduction ofacetylene into the gas stream flowing past the nodules untilmeasurement of the acetylene-induced decline in nitrogenaseactivity, decreased substantially. This was characteristic ofan increase in the permeability of the nodules to gaseous diffusionupon rehydration. However, calculated values of nodule diffusionresistance after the 24 h periods of rehydration did not indicateany recovery of gaseous diffusion resistance based on measurementsof the respiration coupled to nitrogenase activity. Hence, useof a diffusion analogue (i.e. Fick's Law) in conjunction withnodule respiratory CO₂ efflux was unable to predict changesin permeability of the variable barrier of legume nodules followingnodule dehydration and recovery. Key words: Subterranean clover, gaseous diffusion, respiration, carbohydrates, drought     

Changes in Nitrogenase Activity and Nodule Diffusion Resistance of Subterranean Clover in Response to pO2

Diffusion resistance to oxygen within nodules was calculatedusing the respiratory quotient (RQ) of nodules from intact plantsof subterranean clover (Trifolium subterraneum L.) cv. SeatonPark nodulated by Rhizobiun trifolii WU95. From 21 to 52% O₂,the RQ remained between 0.94 and 1.04, whereas at 10% O₂, theRQ was 1.65. When nodulated roots of intact plants were exposedto sub-ambient pO₂ in a continuous flow-through system, respirationdeclined immediately, followed by a partial recovery within30 min. The magnitude of the final respiration rate was dependentupon the pO₂ in the gas stream. Initial rates of respirationwere re-established after 24 h at sub-ambient pO₂ as a resultof changes in the resistance of the variable barrier to oxygendiffusion within the nodules. Nitrogenase activity also decreasedlinearly with decreasing pO₂ in the gas stream, but partialrecovery occurred after 24 h incubation at sub-ambient pO₂.Maximum rates of nitrogenase activity occurred at rhizosphereoxygen concentrations between 21% and 36% O₂. Resistance tothe diffusion of oxygen within the nodules increased at supra-ambientpO₂ and at oxygen concentrations above 36% O₂, resulted in adecrease in both nitrogenase activity and nodulated root respiration.The diffusion resistance of nodules to oxygen increased rapidlyin the presence of either supra-ambient pO₂ or saturating pC₂H₂.Reductions in nodule diffusion resistance either during recoveryfrom exposure to 10% acetylene or to sub-ambient pO₂ occurredmore slowly. It is concluded that subterranean clover is welladapted for maximum nitrogen fixation at ambient pO₂. Key words: Nitrogenase activity, oxygen, subterranean clover, diffusion resistance     

Ontogenetic Changes in Root Nodule Subpopulations of Common Bean (Phaseolus vulgaris L.): I. NITROGENASE ACTIVITY AND RESPIRATION DURING POD-FILLING

Nitrogenase activity is commonly measured on a whole plant basis,or only on parts of the root system. In the present paper, activityin different root nodule subpopulations was followed throughoutreproductive growth, in order to characterize the pod-fillingdecline in nitrogenase activity. Inoculated common bean plantswere grown to maturity under controlled environment conditions.Nitrogenase activity (H₂ evolution in air) and nodule respiration(CO₂ evolution) were measured in three separate zones of theroot system with a non-destructive, open flow, gas-exchangesystem. Nitrogenase activity in the top zone drastically declinedat the initiation of pod-filling, whereas nitrogenase activityin the mid zone was stable during the same period. Hence, thepod-filling decline was limited to a certain nodule subpopulationand not of a systemic type. Nodule respiration showed a similar,but less pronounced pattern. The sharp decline in nitrogenaseactivity was not paralleled in nitrogenase specific activity.Nitrogenase activity is not likely to be limited by the availabilityof oxygen or carbohydrates at the onset of pod-filling becausespecific nodule respiration did not change significantly atthis time. In the top zone, nitrogenase specific activity declinedgradually throughout the measurements, whereas in the mid-partof the root system specific activity peaked and gradually declined2-4 weeks later. The dissimilarities between specific and totalnitrogenase activity were explained by differences in nodulegrowth rates. The data suggest that the oldest nodule populationloses activity at the onset of pod-filling. At the same time,nodules grow and nitrogenase activity increases in younger distalparts of the root system. Estimating total nitrogen fixationin this symbiosis by partial sampling of nodulated root systemsis likely to be very misleading. Key words: Nitrogen fixation, respiration, pod-filling decline, Phaseolus vulgaris, ontogeny     

Defoliation in White Clover: Regrowth, Photosynthesis and N2 Fixation

Single plants of white clover, grown in a controlled environmentand dependent for nitrogen on fixation in their root nodules,were defoliated once by removing approximately half their shoottissue. Their regrowth was compared with the growth of comparableundefoliated plants. Two similar experiments were carried out:in the first, plants were defoliated at 2.5 g, and in the secondat 1.2 g total plant d. wt. Defoliation reduced rate of N₂ fixation by > 70 per cent,rate of photosynthesis by 83–96 per cent, and rate ofplant respiration by 30–40 per cent. Nodule weights initiallydeclined following defoliation as a result of loss of carbohydratesand other unidentified components. No immediate shedding ofnodules was observed but nodules on the most severely defoliatedplants exhibited accelerated senescence. The original rates of N₂ fixation were re-attained after 5–6or 9 d regrowth, with increase in plant size at defoliation.In general, the rate of recovery of N₂ fixation was relatedto the re-establishment and increase of the plant's photosyntheticcapacity. Throughout the growth of both defoliated and undefoliatedplants nodule respiration (metabolism) accounted for at least23 ± 2 per cent of gross photosynthesis. The unit ‘cost’of fixing N₂ in root nodules, in terms of photosynthate, appearedto be unaffected by defoliation, except perhaps for plants veryrecently defoliated. Similarly, the percentage nitrogen contentsof shoot, root and nodules of defoliated plants became adaptedwithin a few days to those characteristic of undefoliated plants. Trifolium repens, white clover, N₂ fixation, defoliation, photosynthesis, respiration     

Nitrogenase activity of two genotypes of Acacia mangium as affected by phosphorus nutrition

The specific nodulation, nitrogenase activity (acetylene reduction) and budgets of carbon allocation to respiration by nodulated roots were examined in two provenances of Acacia mangium Willd. grown in a glasshouse for 17 weeks to investigate the effects of soil phosphorus and genotypes of the host plant on symbiotic nitrogen fixation. Application of phosphorus (0–80 mg P kg^-1 soil) increased specific nodulation (g nodule dry weight g^-1 plant dry weight) of provenance Ma11 by two-fold and the percentage of nodulated root respiration allocated to nitrogenase by 50%, but had no effect on specific activity of nitrogenase or specific respiration coupled with nitrogenase activity. Improved phosphorus nutrition increased the specific nitrogenase activity of provenance Ma9 by 2-fold, the percentage of nodulated root respiration allocated to nitrogenase, and specific nitrogenase-linked respiration by 50%, respectively, but had no effect on the specific nodulation. The percentage of respiration coupled with nitrogenase activity in nodulated root respiration by provenance Ma9 was 60–70% higher than that in provenance Ma11, regardless of phosphorus levels applied. At the optimal level of phosphorus addition (10 mg P kg^-1 soil), provenance Ma9 had a lower dry mass than provenance Ma11. This was accompanied by a lower nodulated root respiration and a higher percentage of nodulated root respiration allocated to nitrogenase activity in provenance Ma9.     

Carbon Costs of Nitrogenase Activity in Legume Root Nodules determined using Acetylene and Oxygen

There is a coupled decrease in respiration and nitrogenase activityof nodules of many legume symbioses induced by exposure to acetylenein the presence of 21% O₂. The respiratory costs of nitrogenaseactivity can be determined directly and distinguished from respiratorycosts for growth and maintenance of roots and nodules, usingthe linear regression of respiration on nitrogenase activity.The regression gradient represents the carbon costs for thetransfer of one pair of electrons by nitrogenase in terms ofmoles CO₂ released per mole of ethylene produced. The interceptof the regression is the growth and maintenance respirationof nodules or nodulated roots. Exposure to acetylene at decreasedor increased oxygen concentrations in the range from 10% to70% resulted in a wider range of values for CO₂ production andnitrogenase activity that fell on the same regression line asvalues obtained during the acetylene-induced decline at 21%oxygen. Oxygen concentrations below 10% increased significantlythe proportion of anaerobic respiration and produced changesin nitrogenase activity not correlated with CO₂ production.Provided that these limits are not exceeded, oxygen-inducedchanges in nodule activity in the presence of acetylene canbe used to measure the efficiency of those symbioses which donot exhibit an acetylene-induced decline at a fixed oxygen concentration. Respiratory cost (moles CO₂/mole ethylene) remained relativelyconstant with plant age for detached pea nodules (2.8), attachednodulated roots of lucerne (2.5) and detached nodulated rootsof field bean (4.2). However, for lucerne and field beans theproportion of total root respiration coupled to nitrogenasedeclined with time. A survey of 13 legume species gave values from 2 to 5 molesCO₂/mole C₂H₄ Rhizobium strain and host-dependent variationsin efficiency were found. Key words: Nitrogenase, Legume root nodules, Respiration, Oxygen     

White Clover N2-Fixation in Response to Root Temperature and Nitrate: II. N2-FIXATION, RESPIRATION, AND NITRATE REDUCTASE ACTIVITY

Established, nodulated white clover plants were transferredto eight tanks of a flowing culture apparatus with solutiontemperatures of 5, 11, 17, and 25 ?C (two tanks per temperature).Shoot temperature and light environment were common to all plants.After 7 d, (10 mmol m^–3) was continuouslysupplied to one tank at each temperature while in the remainingfour tanks (one at each temperature) the plants were completelydependent on nodule N₂-fixation. Plants were randomly selected at intervals during the following14 d period in order to measure root and nodule respirationand acetylene reduction activity (ARA) in a flow-through systemset at the adapted root temperature. Additional plants wereassayed for in vitro nitrate reductase activity in leaves, roots,and nodules. Apparent nitrogenase activity (ARA) and respiration associatedwith it were each markedly affected by temperature in two ways;(1) Activity per unit weight of nodule was reduced at lowertemperatures; (2) Development of the plant, and thus also nodulemass, was restricted at lower temperatures which, in turn, restrictedtotal nodule activity per plant. The presence of nitrate significantly reduced ARA of nodules,particularly at higher temperatures. However, significant discrepancieswere found when N₂-fixation rates, estimated from the acetylenereduction assay, were compared with N₂-fixation rates calculatedfrom curves fitted to N accumulation data (minus the rate of uptake in the case of nitrate-treated plants). Carbon use efficiency (CO² respired per C₂H₄ produced) was notsignificantly affected by temperature or the presence of nitrate. Nitrate reductase activity (NRA) developed in all plant partsat the three highest temperatures, but not at 5 ?C. We calculatethat leaf NRA may account for 82, 75, and 68% of total nitratereduction at 11, 17, and 25 ?C respectively. Key words: Trifolium repens, white clover, N₂ fixation, root temperature, acetylene reduction assay, nitrate, nitrate reductase     

Responses of N2 Fixation-linked Respiration to Host-plant Energy Status in White Clover Acclimated to a Controlled Environment

Ryle, G. J. A., Powell, C. E. and Gordon, A. J. 1988. Responsesof N₂ fixation-linked respiration to host-plant energy statusin white clover acclimated to a controlled environment.—J.exp. Bot. 39: 879–887. Single plants of white clover, acclimated to a controlled environmentand dependent for nitrogen on N₂ fixation in their root nodules,were darkened, defoliated or exposed to enhanced CO₂ levelsto establish the quantitative relationships between the photosynthesisof the host plant and the N₂ fixation metabolism of root nodules. The nodule respiration associated with N₂ fixation (FLR) declinedrapidly to 10–15% of its normal rate following plant darkeningearly in the photoperiod. Darkening at progressively later intervalsduring the photoperiod demonstrated a positive, apparently linearrelationship between duration of illumination and total FLRduring the photoperiod and the following night period. Completeor partial defoliation reduced FLR according to the leaf arearemoved: again, there was a strong positive correlation betweencurrent rate of photosynthesis, whether of defoliated or undefoliatedplants and the FLR of root nodules. Doubling the current rateof photosynthesis, by enhancing CO₂ levels around the shoots,promoted FLR within 1–2 h when plants were stressed bylack of light. However, enhanced CO₂ levels increased FLR onlyslowly over a period of several hours in plants entrained tothe normal growing conditions. It is concluded that, in these plants acclimated to a uniformand favourable controlled environment, the supply and utilizationof photosynthetic assimilate in N₂ fixation was finely balancedand quantitatively linked during a single diurnal period andthat nodule functioning was not depressed by lack of energysubstrate. Key words: White clover, N₂ fixation, photosynthesis.     

N2 Fixation and the Respiratory Costs of Nodules, Nitrogenase Activity, and Nodule Growth and Maintenance in Fiskeby Soyabean

The respiratory effluxes of nodules and of roots of FiskebyV soyabean (Glycine max (L.) Merr.), grown in a controlled environment,were measured at intervals in air and 3% O₂ from shortly afterthe onset of N₂ fixation until plant senescence. The respiratoryburdens linked with nitrogenase plus ammonia metabolism, andnodule growth and maintenance, were calculated from gas exchangedata and related to the concurrent rates of N₂ fixation. The specific respiration rates of nodules increased to a maximumof 21 mg CO₂ g^–1 h^–1 at the time pods began development:the equivalent maximum for roots was c. 4.5 mg CO₂ g^–1h^–1. Maximum nodule and root respiration rates per plantwere attained about 25 d later at the time N₂ fixation peakedat 15 mg N d^–1 plant^–1. The relationship between nodule respiration and N₂ fixationindicated an average respiratory cost of 13.2 mg CO₂ mg^–1N until the last few days of plant development Separation ofnodule respiration into the two components: nitrogenase (+ NH₃metabolism) respiration and nodule growth and maintenance respiration,indicated that the latter efflux accounted for c. 20% of nodulerespiration while N₂ fixation was increasing and new noduletissue was being formed. When nodule growth ceased and N₂ fixationdeclined, this component of respiration also declined. The respiratorycost of nitrogenase activity plus the associated metabolismof NH₃ varied between 11 mg CO₂ mg^–1 N during vegetativeand early reproductive growth, to 12.5 mg CO₂ mg^–1 N duringthe later stages of pod development. Key words: N2 fixation, Respiration, Nodules, Nitrogenase     

Studies on the Relation Between Net Photosynthesis and Nitrogenase-linked Respiration in Subterranean Clover

The relation between the rate of nitrogenase-linked respirationand net photosynthesis, and the effect of defoliation on thisrelation, was studied in plants of subterranean clover (Trifoliumsubterraneum L. cv. Seaton Park). Nitrogenase-linked respirationwas estimated as the difference between the rate of nodulatedroot respiration at 21% O₂ and at 3% O₂. The level to which the rate of nitrogenase-linked respirationfell several hours after defoliation was directly proportionalto the decline in the rate of net photosynthesis. Approximately9% of net photosynthesis was always expended in nitrogenaseactivity, irrespective of whether or not the plants were defoliated.This proportion was maintained during the first 3 d of regrowth. To determine whether the decline in nitrogenase-linked respirationafter defoliation was due solely to the decline in the rateof photosynthesis, a further experiment was conducted in whichthe pre-defoliation rate of net photosynthesis was restoredimmediately (with supplementary light) or within 5 min (supplementarylight and CO₂) after defoliation. Restoring the rate of netphotosynthesis did not prevent the post-defoliation declinein nitrogenase-linked respiration. However, when photosynthesiswas reduced to zero by the imposition of darkness, and the rateof nitrogenase-linked respiration allowed to decline to a steadyrate after 3 h, a rapid recovery in the rate of nodulated rootrespiration began within 2 h of returning the plants to thelight. It was hypothesized that a ‘shoot factor’,which was affected by defoliation, could override the apparentrelation between nitrogenase-linked respiration and the rateof current photosynthesis. Key words: Defoliation, N₂ fixation, photosynthesis, nitrogenase-linked respiration, subterranean clover     

Further Errors in the Acetylene Reduction Assay: Effects of Plant Disturbance

A flow-through gas system was used to study the effects of disturbanceon nitrogenase (acetylene reduction) activity of nodulated rootsystems of soyabean (Glycine max) and white clover (Trifoliumrepens). Detopping plus removal of the rooting medium (by shaking)produced a substantial decrease in maximum nitrogenase activity.This response is due to a reduction in oxygen flux to the bacteroidscaused by an increase in the oxygen diffusion resistance ofthe nodule. The decrease in maximum nitrogenase activity wasmuch smaller for roots subjected to detopping only. Thus, theeffect of root shaking is more important than that of shootremoval. The effect of detopping plus root shaking on nitrogenase activityoccurred whether the plants were equilibrated and assayed at25°C or 15°C. However, the effect of disturbance onthe oxygen diffusion resistance of the nodules, and thus onnitrogenase activity, was greater at the higher temperature.At the lower temperature the oxygen diffusion resistance ofthe nodules had already been increased in response to the reducedrequirement for oxygen. These nodules were less susceptibleto the effects of disturbance. Thus, comparisons of the effectsof equilibration temperature on nitrogenase activity produceddifferent results depending on whether intact or disturbed systemswere used. With intact systems activity was lower at the lowertemperature but with detopped/shaken roots the lowest activityoccurred at the higher temperature. It is concluded that the use of detopped/shaken roots can producesubstantial errors in the acetylene reduction assay, which makesthe assay invalid even when used for comparative purposes. However,comparisons with rates of ¹⁵N₂ fixation and H₂ production showthat accurate measurements of nitrogenase activity can be obtainedfrom maximum rates of acetylene reduction by intact plants ina flow-through gas system. The continued use of assay proceduresin which cumulated ethylene production from disturbed systemsis measured in closed vessels must be questioned. Key words: Nodules, acetylene, nitrogenase activity     

Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules

The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO₂ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O₂. Phloemgirdling was effected 20 hours and exposure to Ar:O₂ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO₂ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O₂ decreased nodule CO₂ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO₂ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to ¹⁴CO₂. In contrast to nodules, roots exported very little radioactivity, and most of the ¹⁴C was exported as organic acids. The nonphotosynthetic CO₂ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO₂ assimilation. Nodules fixed CO₂ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO₂ fixation. The results of this study showed that exposure of nodules to Ar:O₂ reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO₂ fixation, nitrogenase activity, nodule-specific respiration, and transport of ¹⁴C photoassimilate to nodules. These results indicate that nodule CO₂ fixation in alfalfa is associated with N assimilation.     

A Major Error in the Acetylene Reduction Assay: Decreases in Nodular Nitrogenase Activity Under Assay Conditions

Observations on both attached nodulated roots and detached noduleshave revealed that nitrogenase activity in many legume speciesdeclined rapidly in the presence of acetylene, with a concurrentreduction in respiration. The reduction began within a few minutesof exposure to acetylene and continued for 30–60 min beforea new steady-state was attained. A similar decline in H₂ evolutionand respiration was observed when N₂ was replaced with argonor helium. This suggests that the decrease is linked to thecessation of ammonia production. Measurements of ¹⁵N₂ uptakedemonstrated that it is the pre-decline rather than final rateof ethylene production which represents the real rate of nitrogenaseactivity. The implications of these findings for the interpretationof acetylene reduction and hydrogen evolution data are considered. Key words: Roots, Acetylene, Nitrogenase activity     

Role of Oxygen in the Limitation and Inhibition of Nitrogenase Activity and Respiration Rate in Individual Soybean Nodules

Although infected cell O2 concentration (Oi) is known to limit respiration and nitrogenase activity in legume nodules, techniques have not been available to measure both processes simultaneously in an individual legume nodule. Consequently, details of the relationship between nitrogenase activity and Oi are not fully appreciated. For the present study, a probe was designed that allowed open circuit measurements of H2 evolution (nitrogenase activity) and CO2 evolution (respiration rate) in a single attached soybean nodule while simultaneously monitoring fractional oxygenation of leghemoglobin (and thereby Oi) with a nodule oximeter. Compared to measurements of whole nodulated roots, use of the probe led to inhibition of nitrogenase activity in the single nodules. During oximetry measurements, total nitrogenase activity (TNA; peak H2 evolution in Ar/O2) in the single nodules was 16% of that in whole nodulated roots and 48% of nodulated root activity when Oi was not being measured simultaneously. This inhibition did not affect the nodules' ability to regulate Oi, because exposure to Ar/O2 (80:20, v/v) caused nitrogenase activity and respiration rate to decline, and this decline was linearly correlated with a concurrent decrease in Oi. When the nodules were subsequently exposed to a linear increase in external pO2 from 20 to 100% O2 at 2.7% O2/min, fractional leghemoglobin oxygenation first increased gradually and then more rapidly, reaching saturation at a pO2 between 76 and 100% O2. Plots of nitrogenase activity and respiration rate against Oi showed that rates increased with Oi up to a value of 57 nM, with half-maximal rates being attained at Oi values between 10 and 14 nM O2. The maximum nitrogenase activity achieved during the increase in pO2 (potential nitrogenase activity) was 30 to 57% of that measured in intact nodulated roots, showing that O2 limitation of nitrogenase activity could account for a significant proportion of the inhibition of TNA associated with the use of the probe. However, some factor(s) in addition to O2 must have limited the activity of single nodules at both subsaturating and saturating Oi. At Oi values greater than about 57 nM, nitrogenase activity and nodule respiration were inhibited, but, because this inhibition has been shown previously to be readily reversible when the Oi was lowered, it was not attributed to direct O2 inactivation of the nitrogenase protein. These results indicate that maximum nitrogenase activity in legume nodules is supported by a narrow range of Oi values. Possible biochemical mechanisms are discussed for both O2 limitation of nitrogenase activity at low Oi and inhibition of nitrogenase activity at high Oi.     

Relationships Between Nitrate and Oxygen Supply in Symbiotic Nitrogen Fixation by White Clover

Exposure of mature, nodulated plants of white clover (Trifoliumrepens) cv. Blanca to 330 mg dm^–3 NO₃-N for 8 d causednitrogenase activity per plant to decrease by 80%. Total nodulatedroot respiration was not significantly affected but analysisof its components showed an 81% decrease in nitrogenase-linkedrespiration and a 340% increase in growth and maintenance respiration.Carbon costs of nitrogenase activity (mol CO₂ respired per molC₂H₄ produced) increased by 45% over the exposure period. Sucrosecontent of the nodules decreased, but the pattern of decreasedid not correlate with that of nitrogenase activity. The oxygendiffusion resistance of the nodules was increased by a factorof five. Characterization of this resistance increase suggestsan abnormal modification of the diffusion barrier and it isconcluded that alteration in the oxygen supply to the bacteroidsis involved in the effect of nitrate on nitrogenase activity. Key words: Nitrogenase activity, nitrate, oxygen     

Loss and recovery of nitrogenase in Abuts incana nodules exposed to low oxygen and low temperature

The possibility that respiration limits oxygen access to nitrogenase was tested by artificially upsetting the balance between oxygen consumption (respiration) and oxygen influx (diffusion). Argon treatment of the nodulated root system on intact plants stopped in vivo nitrogenase activity almost completely. Upon return to air, nitrogenase activity was very low and recovered gradually to full activity after about 5 h. In vitro measurements on nodule homogenates indicated that active nitrogenase was lost upon the shift from low (argon) to normal (air) oxygen. Maintenance of nodulated root systems at low temperature (2°C) inhibited both respiration and in vivo nitrogenase activity. Upon return to normal temperature (22°C), oxygen uptake recovered very rapidly, but nitrogenase activity recovered only gradually to full activity after about 5 to 6 h. Again, loss of active nitrogenase could, at least partly, explain the reduced in vivo nitrogenase activity. The effects from a temporarily impaired balance between oxygen consumption and oxygen influx thus point to the importance of respiration for limiting oxygen access to nitrogenase.     

Effect of Temperature on Nitrogenase Activity in White Clover

Single, clonal plants of white clover were grown without inorganicnitrogen in four contrasting day/night temperature regimes,with a 12 h photoperiod, in controlled environments. Root andnodule respiration and acetylene reduction activity were measuredin a flow-through system during both day and night for plantsacclimated to day/night regimes of 23/18, 15/10 and 10/5 ?C.Similar measurements were made on plants acclimated to 20/15?C and stepwise at temperatures from 4 to 33 ?C. Peak rate of ethylene production, nitrogenase-linked respirationand basal root + nodule respiration increased approximatelylinearly from 5 to 23 ?C both in temperature-acclimated plantsand in plants exposed to varying measurement temperatures. Themeasured attributes did not vary significantly between day andnight. Temperatures above 23–25 ?C did not further enhancethe rate of ethylene production, which remained essentiallythe same up to the maximum measured temperature of 33 ?C. The measurements of nitrogenase-linked respiration between 5and 23 ?C, during both day and night, demonstrated a constant‘energetic cost’ of acetylene reduction of 2.9 µmolCO₂ µmol C₂H₄^–1,. Over the same temperature range,the approximate activation energy of acetylene reduction was60 kJ mol^–1. The integrated day plus night nitrogenase-linkedrespiration accounted for 13.4–16% of the plant‘snet shoot photosynthesis in a single diurnal period: there wasno significant effect of temperature between 5 and 23 ?C. Key words: Trifolium repens, white clover, temperature, N₂ fixation, respiration