Oxygen Supply Limits Nitrogenase Activity of Clover Nodules After Defoliation

The aim of this study was to test the effect of oxygen partialpressure as a possible limiting factor of nitrogen fixationfollowing defoliation. The response of nitrogenase activity(C₂H₂-reduction) of defoliated and undefoliated white and redclover plants (Trifolium repens L. and Trifolium pratense L.)to either 19 kPa oxygen or 55 kPa oxygen was investigated. Priorto defoliation, white clover plants were grown for five weeksin a growth chamber, and red clover plants for 7 or 11 weeksin a glasshouse. The results included measurements of ¹⁶N₂-uptake. Increasing oxygen partial pressure from 19 to 55 kPa severelyrestricted nitrogenase activity of undefoliated white cloverplants; however, 2 h after complete defoliation, the same treatmentcaused a significant increase. A fivefold increase in nitrogenaseactivity upon exposure to the elevated oxygen partial pressurewas found at the end of a 24 h period. This beneficial effectdecreased gradually from 1 to 5 d after defoliation. The responseof recently defoliated red clover plants to 55 kPa oxygen partialpressure was similar to that of white clover, independentlyof plant age. The gradual recovery of nitrogenase activity duringthree weeks of regrowth was associated with a simultaneous changein the response to increased oxygen partial pressure, leadingagain to the response of undefoliated plants. These data suggested that lack of oxygen at the site of nitrogenfixation, resulting from a dramatic increase in oxygen-diffusionresistance, is the main factor limiting nitrogenase activityfollowing defoliation. Trifolium repens L., Trifolium pratense L., white clover, red clover, defoliation, regrowth, nodules, nitrogen fixation, nitrogenase activity, oxygen limitation     

Acetylene reduction assay on white clover genotypes and on grass/clover swards

Acetylene reduction assay was used to measure the nitrogenase activity of white clover genotypes in pots and of grass/clover swards in situ. Much of the variation in nitrogenase activity of single genotypes and hybrid populations was associated with plant dry weight. After adjustment for plant dry weight it was concluded that there was limited scope for selection for increased nitrogenase activity. In plant breeding this technique would seem to have greatest application in the selection for continued nitrogen fixation activity in the presence of inhibitory factors such as high levels of mineral nitrogen. The in situ studies revealed differences in nitrogenase activity of grass/clover swards based on contrasting cultivars of white clover. These differences were due to variation in clover density and also to variation in activity per unit clover dry weight. It was concluded that the variation in nitrogenase activity per unit dry weight reflected differences in growth pattern of the cultivars in the autumn when these assays were made. The in situ studies offer a means whereby the nitrogenase activity of cultivars and selected families can be monitored under varying levels of mineral nitrogen and other husbandry treatments without disruption of the sward.     

The Influence of Carbohydrate Reserves on the Response of Nodulated White Clover to Defoliation

A growth-chamber study was carried out to determine whetherthe response of apparent nitrogenase activity (C₂ H₂ reduction)to complete defoliation is influenced by the availability ofcarbohydrate reserves Reserve carbohydrate (TNC) concentrationsof 6-week-old white clover (Trifoliun repens L) plants weremodified by CO₂ pretreatments There was no difference in theresponse of apparent nitrogenase activity to defoliation betweenplants with different TNC concentrations C₂H₂ reduction activitydeclined sharply after defoliation and then recovered similarlyin both high- and low-TNC plants Further experiments were conductedto explain the lack of response of apparent nitrogenase activityto TNC levels Bacteroid degradation was ruled out because invitro nitrogenase activity of crude nodule extracts was stillintact 24 h after defoliation Sufficient carbohydrates appearedto be available to the nodules of defoliated plants becauseadding [¹⁴C]glucose to the nutrient solution did not preventthe decline in apparent nitrogenase activity These conclusionswere supported by the finding that an increase in pO₂ aroundthe nodules of defoliated plants completely restored their C₂H₂reduction activity The comparison of the effects of defoliationand darkness suggested that the decrease in apparent nitrogenaseactivity was not related directly to the interruption of photosynthesisIt appears that lack of photosynthates is not the immediatecause of the decline of nitrogen-fixing activity after defoliation White clover, Trifolium repens L, defoliation, nitrogen fixation, regrowth, reserves, carbohydrates, acetylene reduction, nodule extract     

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.     

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     

Assimilate Partitioning in Red and White Clover Either Dependent on N2 Fixation in Root Nodules or Utilizing Nitrate Nitrogen

During vegetative growth in controlled environments, the patternof distribution of ¹⁴C-labelled assimilates to shoot and root,and to the meristems of the shoot, was measured in red and whiteclover plants either wholly dependent on N₂ fixation in rootnodules or receiving abundant nitrate nitrogen but lacking nodules. In experiments where single leaves on the primary shoot wereexposed to ¹⁴CO₂, nodulated plants of both clovers generallyexported more of their labelled assimilates to root (+nodules),than equivalent plants utilizing nitrate nitrogen, and thiswas offset by reduced export to branches (red clover) or stolons(white clover). The intensity of these effects varied with experiment.The export of labelled assimilate to growing leaves at the terminalmeristem of the donor shoot was not influenced by source ofnitrogen. Internode elongation in the donor shoot utilized nolabelled assimilate. Whole plants of white clover exposed to ¹⁴CO₂ on seven occasionsover 32 days exhibited the same effect on export to root (+nodules),which increased slightly in intensity with increasing plantage. Nodulated plants had larger root: shoot ratios than theirequivalents utilizing nitrate nitrogen. Trifolium repens, Trifolium pratense, red clover, white clover, nitrogen fixation, nitrate utilization, assimilate partitioning     

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     

Effects of Temperature on Growth and Nitrogen Fixation by Swards of Subterranean Clover

Subterranean clover (Trifolium subterraneum L.) cv. ‘Woogenellup’ swards were grown at 10, 15, 20 and 25 Cwith a 12 h photoperiod of 500 or 1000 µmol m^–2s^–1 [low and high photosynthetic photon flux density (PPFD)].Nitrogen-fixing swards received nutrient solution lacking combinednitrogen while control swards received a complete nutrient solution.Growth was measured by infra-red analysis of carbon dioxideexchange and by accumulation of dry matter. Swards were harvestedat intervals between 95 and 570 g d. wt m^–2 for estimationof nitrogenase activity by acetylene reduction and hydrogenevolution assays. Nitrogen fixation was also measured by increasein organic nitrogen. The growth rate was highest at 10 C at low PPFD, and at 10–15C at high PPFD. Nitrogen-fixing swards grew slower than thosereceiving combined nitrogen. Nitrogen fixation measured by increasein organic nitrogen responded similarly to the growth rate,as did acetylene reduction between 10 and 20 C. At 25 C therelationship between acetylene reduction and nitrogen fixationwas distrupted. The difference between the rates of acetylenereduction and hydrogen evolution, theoretically proportionalto nitrogen fixation, was not a reliable indicator of nitrogenfixation because hydrogen uptake developed. Trifolium subterraneum L, subterranean clover, growth, nitrogen fixation, temperature, acetylene reduction     

Oxygen, the NifA gene, Nodule Structure and the Initiation of Nitrogen Fixation

Both nitrogenase and the NifA gene, which controls the expressionof the nitrogenase enzyme, require microaerobic conditions inthe infected zone of a nodule. Such conditions depend on theability of the respiratory system to consume oxygen at approximatelythe maximum rate at which it can enter that region by diffusion.The balance between consumption and supply governs the minimumsize of a functional nodule. The nature of this balance is exploredin this paper using a model. It shows that it appears physicallyimpossible for legume root nodules, below a certain minimumsize, to fix nitrogen. Furthermore, experimental data are usedto suggest that the alternative oxidase respiratory systemsmay initially provide the large respiratory capacity requiredto create microaerobic conditions, suitable for the NifA geneand nitrogenase. Nitrogen fixation, NifA gene, diffusion resistance, oxygen     

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     

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 Influence of Irradiance Distribution on the Growth of White Clover (Trifolium repens L.) in Differently Managed Canopies of Permanent Grassland

Growth of white clover was investigated in permanent grasslandcut three or five times per year. The influence of cutting frequencyand nitrogen fertilization on dry-matter yield, leaf-area distributionand the distribution of photosynthetically active radiationwithin the canopy were examined. In the five cut treatments, total dry-matter yield was nearlyequal, with and without nitrogen. However, nitrogen practicallyeliminated white clover. Leaf-area distribution showed characteristicpatterns for the different treatments. The small proportionof white clover in the treatment with nitrogen fertilizationwas thought to be due to the large leaf area of the other speciesat heights which white clover could not attain. This conclusionwas supported additionally by the radiation measurements withinthe canopy. The sunlit fractional area within canopy layers was measuredwith ‘quantum sensors’ and calculated from canopytransmission measured with tube solarimeters. The leaf areaindex of white clover was highly correlated (r² = 0.68) withthe sunlit fractional area above the canopy layers where whiteclover was present. This response of white clover leaf growth to the light regimeis discussed in relation to the potential petiole growth. White clover, Trifolium repens L., permanent grassland, irradiance distribution, sunlit fractional area, petiole extension, leaf area, dry matter, stratified clipping     

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     

Effects of some surfactant fungicides on Rhizobium trtfolii and its symbiotic relationship with white clover

Effects of the surfactant apple mildew eradicants Manoxol OT, Alk 3, Triton X45, PP 222, Ethylan CP, Hyamine 3500, Aromox DMMCDW and Aromox C12W on growth and respiration of R. trifolii in vitro and on its symbiotic nitrogen fixation in association with white clover were measured. Most treatments reduced growth up to 8 wk; after 12 wk plant weight and nitrogenase activity were significantly reduced (P = 0.01) by Triton X45, Manoxol OT and PP 222 at concentrations of 1000, 2500 and 5000 μg/gram of soil respectively. Electron microscopy showed that bacteroids in root nodules where nitrogen fixation was inhibited had contracted away from the host membrane leaving an intracellular space. Damage to the bacteroid membrane was apparent. Surfactants had no demonstrable effect on adhesion of the bacteria to root hairs. In apple orchards run-off of surfactant fungicides, applied to apple trees at the recommended rates is unlikely to have adverse effects on Rhizobium nodulated white clover.     

The physiology and biochemistry of cultivar-strain interactions in the white clover-Rhizobium symbiosis

Two white clover cultivars were inoculated with two Rhizobium leguminosarum bv. trifolii strains in a factorial series of experiments. Plants were grown in axenic conditions in nitrogen free nutrient solution in a controlled environment room. Variations in nitrogen fixation were dependent partly upon general strain effects, partly upon general cultivar effects but there were also substantial differences attributable to precise interactions between specific combinations. The physiological and biochemical basis of these differences was examined. There were variations in the onset of nodulation and nitrogenase (acetylene reduction) activity. The rate at which nitrogenase activity developed also differed between associations as did the average size and number of nodules but none of these effects correlated well with differences in plant dry matter accumulation. Studies on nodule biochemistry revealed that the major nitrogen fixation enzymes were present in all four associations. Nodule protein content and enzyme activity (on a g nodule fresh weight basis) were substantially greater in associations formed by the more effective strain but cannot explain the interactive effect on dry matter accumulation. The relevance of these data to our understanding of factors regulating variations in nitrogen fixation is discussed.     

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     

Effects of Acetylene and External Oxygen Concentration on the Respiratory Quotient (RQ) of Nodulated Roots of Soyabean and White Clover

Carbon dioxide output and oxygen uptake by intact nodulatedroots of soyabean and white clover were determined over a rangeof external oxygen concentrations (10 to 60%) in the presenceand absence of 10% acetylene. The responses of both sets ofroot systems were in agreement with the known characteristicsof the variable oxygen diffusion resistance of the nodules.These measurements were used to calculate the respiratory quotient(RQ, CO2/O2) for both total root respiration and nitrogenase-linkedrespiration values for each external oxygen concentration. Inthe absence of acetylene, the RQ values of soyabean total rootrespiration showed no significant response to external oxygen,with a mean of 1.06, but differences in values for white cloverroots were significant at P=0.05, with a mean of 1.09. The sametrend was shown for RQ values of nitrogenase-linked respiration,with mean values of 1.10 and 1.12 for soyabean and clover, respectively.The addition of acetylene caused a small but significant initialreduction in RQ of total root respiration with both soyabeanand white clover, as did the subsequent reduction of externaloxygen to 10%. For white clover, increases in external oxygenconcentration to 50% and 60% also produced significant increasesin the RQ of total root respiration compared to that in air.These variations were more pronounced for RQ values of nitrogenaselinkedrespiration. Results are discussed in relation to the use ofRQ values of unity in calculations of nodular oxygen diffusionresistance and the involvement of physical and biochemical processesin the regulation of oxygen supply to bacteroids. Key words: Respiratory quotient, nodules, oxygen, diffusion resistance     

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.     

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