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     

A New Procedure for the Calculation of Oxygen Diffusion Resistance in Legume Nodules from Flow-through Gas Analysis Data

Plants of white lupin (Lupinus albus L cv Multolupa) and soyabean(Glycine max L cv Clarke) were grown in controlled-environmentcabinets, subjected to various stresses and their nodular nitrogenaseactivity and total root respiration measured When these measurementswere used to calculate nodular oxygen diffusion resistance,using a simplified equation for Fick's first law of diffusion,it was found that the apparent resistance of stressed nodulesincreased anomalously with decreases in external oxygen concentrationA new analysis procedure is proposed to alleviate this anomalyThis procedure also uses the simplified Fick's law equationbut includes a respiratory contribution to the total oxygenflux across the diffusion barrier which is not coupled to nitrogenaseactivity Also, resistance is modelled as an exponential functionof external oxygen concentration Use of this analysis procedureproduces realistic values for total resistance and providesa characterisation of this resistance into a minimum value andan adjustment factor for changes in external oxygen It is postulatedthat the additional respiration component represents the activityof nodule cortex cells involved in the diffusion barrier, particularlythat of vascular bundles Oxygen diffusion resistance, nodule, nitrogen fixation, respiration     

Characterization of the Resistance to Oxygen Diffusion in Legume Nodules

A method for characterizing the resistance to oxygen diffusionin legume nodules has been developed. This is based on the assumptionsthat diffusion can be described using a simple resistance analogueand that the respiratory response of the bacteriod-containingcells to external oxygen concentrations can be analysed as adiffusion-limited process. Applying this analysis to experimentaldata from infact white clover plants allowed the total diffusionresistance to be separated into (a) a minimum resistance and(b) the extent to which this resistance can be increased. Whenthe carbohydrate status of the nodules was reduced by dark treatments,the minimum diffusion resistance increased, and after 24–28h darkness equalled the maximum resistance. At the same timethe ability to control this resistance was lost. White clover, nitrogen fixation, oxygen diffusion, nodule respiration     

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     

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.     

The Respiratory Costs of Nitrogen Fixation in Soyabean, Cowpea, and White Clover: II. COMPARISONS OF THE COST OF NITROGEN FIXATION AND THE UTILIZATION OF COMBINED NITROGEN

Plants of soyabean, cowpea, and white clover were grown singlyin pots in Saxcil growth cabinets at 23/18 °C, 30/24 °C,and 20/15 °C, respectively, until seed maturation or for85 d (white clover). Two populations were produced within eachspecies: one population effectively nodulated and wholly dependentfor nitrogen on fixation in the root nodules, and a second populationcompletely lacking nodules but receiving abundant nitrate nitrogen.In each species, the two populations were compared in termsof rate of gross photosynthesis, rate of shoot respiration,and rate of root respiration. Source of nitrogen had littleor no effect on rate of photosynthesis or shoot respiration.In contrast, the rate of respiration of the nodulated rootsof plants fixing their own nitrogen was greater, sometimes two-foldgreater, than that of equivalent plants lacking nodules andutilizing nitrate nitrogen. This superiority in terms of rateof root respiration was generally confined to the period ofintense nitrogen fixation. An analysis of the magnitude of thisrespiratory burden in terms of daily photosynthesis indicatesthat, in all three legumes, plants fixing their own nitrogenrespire 11–13% more of their fixed carbon each day thanequivalent plants lacking nodules and utilizing nitrate nitrogen.     

Biological Control of the Resistance to Oxygen Flux in Nodules

Experiments have been conducted to investigate the responseof symbiotic nitrogen fixation in white clover to differentconcentrations of oxygen around the root. Over a wide rangeof oxygen concentrations (21–80 per cent) there was littlevariation in respiration and no evidence of damage to nitrogenase.It is suggested that, in the absence of respiratory protection,oxygen damage to nitrogenase is prevented by changes in theresistance to gaseous diffusion. Trifolium repens L, white clover, nitrogen fixation, nitrogenase, oxygen flux     

The effect of waterlogging on nitrogen fixation and nodule morphology in soil-grown white clover (Trifolium repens L.)

Nodulated, pot-grown plants of white clover (Trifolium repenscv. Katrina) were subjected to different soil moisture regimesand the effect of these treatments on dry matter production,nitrogenase activity, aerenchyma formation, and bacteroid distributionwas determined. In the first experiment, after 9 weeks growthshoots were significantly (P<0.01) heavier for clover plantswhich were flooded from germination compared with those subjectedto normal watering, indicating that clover can adapt to long-termwaterlogging. In the second experiment, time-courses of acetylenereduction showed a lag phase of only 10 min for waterloggedplants, suggesting that gas exchange was occurring through aerationpathways rather than the water. Flooded plants that were drained24 h prior to acetylene reduction assays had substantially greaternitrogenase activity than normally watered or continually floodedplants. However, there was a marked decrease in nitrogenaseactivity when plants which had been watered normally were flooded,suggesting a sensitivity of white clover to sudden changes inmoisture conditions. Morphological studies of nodules from plantsgrown in normally watered and continually flooded soil showedincreased aerenchyma production around roots and nodules ofwaterlogged plants. In addition, the infected cells of submergednodules were larger and had larger vacuoles than those fromnodules of normally watered plants. This increase in vacuolevolume to protoplast volume in infected cells may play a rolein the tolerance of white clover nodules to waterlogging. Key words: White clover, waterlogging, hypoxia, nitrogen fixation, root nodule morphology.     

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     

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     

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     

The Physiology and Nitrogen-fixing Capability of Aquatically and Terrestrially Grown Neptunia plena: The Importance of Nodule Oxygen Supply

The aquatic legume Neptunia plena (L.) Benth. was grown in non-aeratedwater culture or vermiculite. Growth, nodulation, nitrogen fixationand nodule physiology were investigated. Over an 80-d period,plants grew and fixed nitrogen and carbon equally well in bothrooting media, although distribution of growth between plantparts varied. Total nodule dry weights and volumes were similarbut vermiculite-grown plants had three times as many (smaller)nodules than those grown in water. Oxygen diffusion resistanceof nodules exposed to 21% oxygen and 10% acetylene did not differsignificantly. Both treatments showed similar declines in rootrespiration and acetylene reduction activity (approx. 10%) whenroot systems were exposed to stepped decreases and increasesin rhizosphere oxygen concentration. However, nitrogenase activityof aquatically grown plants was irreversibly inhibited by rapidexposure of nodules to ambient air, whereas vermiculite-grownplants were unaffected. Aeration of water-cultured N. plenareduced stem length (but not mass) and number of nodules perplant. The concentration of nitrogen fixation by 163%. PossibleO₂ transport pathways from the shoot atmosphere to roots andnodules are discussed. Aquatic legume, diffusion resistance, Neptunia plena, nitrogen fixation, oxygen, root nodules     

Defoliation-Induced Stress in Nodules of White Clover: I. CHANGES IN PHYSIOLOGICAL PARAMETERS AND PROTEIN SYNTHESIS

Nodule function and protein synthesis were studied in defoliationstressed white clover plants. Uncut control plants (C) werecompared with plants from two defoliation treatments: (1) continuousdefoliation (CD) where all leaves and petioles were removedeach day; and (2) defoliated/recovered (DR) where, after removalof all leaves and petioles, new leaves were then allowed toregrow. After a single defoliation N₂ fixation (acetylene reductionactivity) and nitrogenase-linked respiration declined by morethan 80% within 3 h and by nearly 100% by 24 h. DR plants beganto fix nitrogen again at a very low level 3 d later and thereafterrose to control levels by 15 d. Continuously defoliated plantsnever recovered N₂ fixation capacity. Nodule protein complementwas assessed by polyacrylamide gel electrophoresis. Major changesoccurred in buffer soluble protein band patterns by 6 d in CDplants, but few changes were evident in SDS soluble proteins.By 9 and 14 d significant disruption of all proteins was evident.The prominent host plant protein, leghaemoglobin (Lb) had disappearedby 14 d. In DR plants the intensity of staining was reducedbut no major changes in band patterns were evident and by 21d nodules were rejuvenated. [³⁵S]-labelled methionine was incorporated into nodule proteinsfrom all treatments throughout the experiment. However, continuousdefoliation caused increasing variability between replicatesin the labelled band patterns. By 21 d CD, much of the labelledprotein was present as amorphous low M_r material which suggestseither disruption of the protein synthesizing machinery or rapidhydrolysis by proteolytic enzymes. Surprisingly [³⁵S]-methionine was never found in Lb from nodulesof any treatment. It is possible that white clover Lb does notcontain any methionine residues or that no synthesis of Lb occurred. Key words: Trifolium repens, white clover, defoliation, protein synthesis, nodules     

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     

The Growth and Photosynthesis of Seedling Plants of White Clover at Low Temperature

The growth of white clover (Trifolium repens L.) in conditionstypical of April in Southern England (8 °C day/4 °Cnight, 12 h photoperiod of 90 J m^–2 s^–1 visibleradiation) was extremely slow, whether the plants were dependentfor nitrogen on fixation by their root nodules or were suppliedwith abundant nitrate; although growth was slower in the nodulatedplants. The reasons for slow growth were a large root: shootratio and a small leaf area, particularly in the nodulated plants,and a low photosynthetic rate in all plants. The probable effectsof these characteristics on the growth of white clover withgrasses in mixed pastures are discussed. Trifolium repens L, white clover, low temperature, leaf area, photosynthetic rate, nitrogen supply, growth     

The Respiratory Costs of Nitrogen Fixation in Soyabean, Cowpea, and White Clover: I. NITROGEN FIXATION AND THE RESPIRATION OF THE NODULATED ROOT

Soyabean, cowpea, and white clover, inoculated with effectiverhizobia, were grown singly with a standard mineral nutritionand light regime in controlled environments until seed maturation(in soyabean and cowpea) or late vegetative growth (white clover).Day/night temperature regimes were 23/18, 30/24, and 20/15 °Cin soyabean, cowpea, and white clover, respectively. The respiratorylosses of CO₂ from the nodulated root systems were studied inrelation to the concurrent rate of fixation of atmospheric nitrogen.Despite differences in development, the effectiveness of thesymbioses, and the temperature of growth, all three legumesexhibited similar respiratory losses from nodulated roots perunit of nitrogen fixed. During intense nitrogen fixation, theaverage respiratory losses for the three legumes varied between6·3 and 6·8 mg C mg^–1 N; within each species,the losses varied more widely at different stages of development.These respiratory burdens reflect the total cost to the plantof the nodule/nitrogen fixation syndrome including the subtendingroots. The results are discussed in relation to the respiratoryeffluxes from nodules and roots, and to biochemical investigationsof the costs of nitrogen fixation.     

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 perenneL.) cv. S23 and White clover (Trifolium repens L.) cv. S100were grown from seed under a constant 20 °C day/15 °Cnight temperature regime and harvested at intervals over and88 d growht period. The swards received a nutrient solutiondaily, which was either High (220 mg l^–1) or Low (10 mgl^–1) in nitrate N. The nitrate was labelled with the ¹⁵Nisotope. An acetylene reduction assay was carried out on eachsward just prior to harvest. Rates of acetylene reduction agreed qualitatively with the ^l5Nanalyses but absolute values did not match (assuming a 4:1 C₂H₄:N₂ratio) and errors in the acetylene assay are discussed. In theLow-N swards clover relied almost entirely on symbioticallyfixed N₂, fixing more than ten times as much as the High-N cloverplants. In the Low-N treatment the grass was N-deficient despiteobtaining much more nitrate per unit root dry weight than clover.In the High-N swards, however, clover took up more nitrate perunit root weight than grass. The High-N clover plants also fixedsome N₂ and maintained a higher total-N content than grass throughoutthe period. There was no evidence of transfer of symbioticallyfixed N from the clover to the grass in either treatment. Trifolium repens, Lolium perenne, nitrate, nitrogen fixation, ¹⁵N, acetylene reduction     

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     

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     

Regrowth by Swards of Subterranean Clover after Defoliation. 2. Carbon Exchange in Shoot, Root and Nodule

The carbon economy of subterranean clover swards subjected tothree defoliation treatments (removal of 30, 70 and 80% of shootdry weight) was compared with that of uncut swards. Carbon dioxideexchange in shoots and roots was measured independently 0, 4,8 and 12 d after defoliation. The respiration linked to nitrogenaseactivity was estimated by comparing root respiration measuredin an atmosphere containing 3% oxygen with the respiration in21% oxygen. Net photosynthesis fell by up to 100% immediately after defoliation.There was a decline of over 60percnt; in root respiration bythe end of the first light period, composed of a rapid declineof 70% in nitrogenase-linked respiration in all treatments anda slower decline of nearly 40% in root plus nodule growth andmaintenance respiration in the more severe treatments. Recoveryof net photosynthesis to rates achieved by uncut swards occurredover 4 d in the 30% cut treatment and at least 12 d in moresevere treatments. Whilst recovery of photosynthesis was theprinciple determinant of recovery of net positive carbon balance,the early reduction in respiration facilitated this outcome.After the immediate decline in nitrogenase-linked respiration,recovery in this component of respiration appeared to be linkedwith the recovery in net photosynthesis (approximately 10% ofnet photosynthesis). Carbon budgets revealed priorities in allocation towards leafin the first 5 d and later also towards root growth in severelydefoliated swards. Root respiration comprised a large respiratorycost (up to 75% of net photosynthesis) during early regrowth. Carbon budget, defoliation, N₂ fixation, photosynthesis, regrowth, respiration, subterranean clover, Trifolium subterraneum L