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     

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     

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     

Partitioning and Utilization of Net Photosynthate in a Nodulated Annual Legume

The economy of carbon in nodulated white lupin (Lupinus albusL.) was studied in terms of consumption of net photosynthatein nitrogen fixation, in maintenance of respiration, and inthe production of dry matter and protein. Net photosynthesisrose to a maximum in early fruiting and then fell abruptly dueto shedding of leaves. Nodulated roots acquired translocateequivalent to 51% of the plant's net photosynthate, 78% of thecarbon of this translocate being respired, 10% entering drymatter, and 12% returning to the shoot attached to productsof nitrogen fixation. Nodules utilized 4?0–6?5 g C infixing 1 g nitrogen. Photosynthate was utilized most effectivelyfor nitrogen fixation in late vegetative growth. Fruits sequestered16% of the plant's net photosynthate, shoot night respiration17%, and dry matter formation in shoot vegetative parts 22%.Averaged over growth, 9?9 g net photosynthate was required toproduce 1 g seed dry matter and 31 g net photosynthate to produce1 g seed protein. Budgets for utilization of the carbon of netphotosynthate were constructed for 10 d intervals of the plant'sgrowth cycle. Feeding of shoots with ¹⁴CO₂ resulted in radiocarbonbecoming partitioned approximately as predicted by these budgets.The dependence of root respiration on recent photosynthate wasassessed by following the time course of release of ¹⁴CO₂ tothe rooting medium of the ¹⁴CO-labelled plants.     

Effectiveness of Lotus Root Nodules: III. EFFECT OF COMBINED NITROGEN ON NODULE EFFECTIVENESS AND FLAVOLAN SYNTHESIS IN PLANT ROOTS

When grown in a nutrient solution containing combined nitrogen(NH₄NO₃), Lotus pedunculatus and L. tenuis seedlings inoculatedwith a fast-growing strain of Rhizoblum (NZP2037) did neitherdevelop root nodules nor develop flavolans in their roots. Incontrast, the roots of nodulated seedlings growing in a nitrogen-freenutrient solution contained flavolans. Flavolan synthesis coincidedwith root nodule development on these plants. When added as a single dose, high concentrations of NH₄NO₃ (5and 10 mg N per plant) stimulated the growth of L. pedunculatusplants but suppressed nodulation and nitrogen fixation. In contrastthe continued supply of a low concentration of NH₄NO₃ (1?0 mgN d^–1 per plant) stimulated nitrogen fixation by up to500%. This large increase in nitrogen fixation was associatedwith a large increase in nodule fresh weight per plant, a doublingof nodule nitrogenase activity, and a lowering of the flavolancontent of the plant roots. The close relationship between nitrogendeficiency, nodule development, and flavolan synthesis in L.pedunculatus meant that it was not possible (by nitrogen pretreatmentof plants) to alter the ineffective nodule response of a Rhizobiumstrain (NZP2213) sensitive to the flavolan present in the rootsof this plant.     

The Effect of Defoliation on Carbohydrate, Protein and Leghaemoglobin Content of White Clover Nodules

Single white clover plants grown in pots of Perlite in a controlledenvironment and completely dependent on N₂ fixation were defoliatedto various degrees (46–85 per cent of shoot weight removed).The soluble protein content of nodules declined by about 20per cent and leghaemoglobin content by 50 per cent in the first4–7 d after defoliation but increased again to controllevels as new leaf tissue appeared. In the short term (2–3h) carbohydrate content of nodules declined to different extentsdepending on the severity of defoliation. The initial declinein N₂ fixation and the respiration associated with it, appearednot to be related to the instantaneous carbohydrate contentof nodules but rather to the supply of current photosynthatefrom the shoot. After 24–48 h, however, the carbohydratecontent of nodules had declined to low levels, regardless ofthe severity (46 or 71 per cent shoot removed) of defoliation.As new leaf tissue appeared carbohydrate levels in all partsof the plant gradually recovered towards control levels. Microscopic examination of nodule sections indicated that onlyafter very severe defoliation (80–85 per cent shoot removed)was nodule deterioration evident. Even here, as the plant establishednew leaves, the damage to nodules was repaired and no noduleloss was apparent. Trifolium repens, white clover, defoliation, carbohydrate, protein, leghaemoglobin     

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     

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     

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     

Partitioning and Utilization of Carbon and Nitrogen in Nodulated Roots and Nodules of Chickpea (Cicer arietinum) Grown at Two Moisture Levels

The partitioning and utilization of carbon (C) and nitrogen(N) in nodulated roots and nodules of chickpea (Cicer arietinumL.) was studied at two moisture levels at 10-d intervals 40–140d after sowing (DAS). More C was used in respiration and lessin growth of nodulated roots and nodules under water stresscompared to controls during all growth stages except at theearly vegetative stage. Similarly, less nitrogen was investedin dry matter of both nodules and nodulated roots under stress,except during the vegetative stage where more nitrogen was used.Calculated over the entire growth period, as much as 14 and20% of the total nitrogen and 3 and 4% of the total carbon fixedby the plant was lost to the rooting medium under controlledand stressed conditions, respectively. The efficiency of nitrogen fixation with respect to net C utilizationwas maximal during seed filling under both control and stressconditions However, the efficiency of nitrogen fixation wasalways greater under drier conditions. Carbon, chickpea (Cicer arietinum L.), nitrogen, nitrogen fixation, partitioning     

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     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 1. Development and Application of 13CO2 Assimilation System at a Constant 13C Abundance

A long-term, steady-state ¹³CO₂ assimilation system at a constantCO₂ concentration with a constant ¹³C abundance was designedand applied to quantitative investigations on the allocationof photoassimilated carbon in nodulated soya bean (Glycine maxL.) plants. The CO₂ concentration in the assimilation chamberand its ¹³C abundance were maintained constant with relativevariances of less than ±0.5 per cent during an 8-h assimilationperiod. At the termination of 8-h ¹³CO₂ assimilation by plantsat early flowering stage, the currently assimilated carbon relativeto total tissue carbon (measured by the degree of isotopic saturation)were for young leaves (including flower buds), 13.9 per cent;mature leaves, 15.7 per cent; stems+petioles, 5.9 per cent;roots, 5.4 per cent and nodules, 6.9 per cent, 48 h after theend of the ¹³CO₂ assimilation period, they were 12.3, 7.5, 7.4,6.8 and 6.1 per cent, respectively. The treatment with a highconcentration of nitrate in the nutrient media significantlydecreased the allocation of ¹³C into nodules. Experiments on¹³CO₂ assimilation by plants at the pod-filling stage were alsoconducted. Labelling by ¹³C was weaker than at the early floweringstage, but an intense accumulation of ¹³C into reproductiveorgans was observed. Glycine max L., nodulated soya bean plants, ¹³CO₂ assimilation, carbon dynamics     

Compartmental Analysis of the Partitioning of Photo-assimilated Carbon in Nodulated Soybean Plants during the Light Period

Kouchi, H., Yoneyama, T. and Akao, S. 1986. Compartmental analysisof the partitioning of photo-assimilated carbon in nodulatedsoybean plants during the light period.—J. exp. Bot. 37:994–1005. Dynamics of the partitioning of photo-assimilated carbon invegetative nodulated soybean (Glycine max L.) plants in thelight period was investigated by compartmental analysis basedon data from steady-state ¹³CO₂ assimilation experiments. Themodel assumes a total of 18 compartments consisting of activeand temporary storage pools for soluble materials, starch andstructural materials in leaves, stems plus petioles, roots andnodules together with respired carbon from the roots and nodules.Carbon flow between compartments was described by 22 rate parameters.The rate parameters were evaluated by a non-linear least squaresearch method to optimize the fitness of the simulated resultswith the experimental tracer distribution. The compartment model was well applicable to interpret the carbonpartitioning in whole plants. The analysis showed that: (I)The largest carbon flux during the light period was to storagematerials (starch and temporary storage soluble pools) in theabove-ground parts. The total flux to storage pools was considerablylarger than the transporting flux to below-ground parts. (2)The main carbon flux to the nodules was via direct phloem pathwaysfrom the shoot and not via the compartment of root soluble materials.This flux was 72% of the total carbon flux from the shoot tothe nodulated root system. (3) A large amount of carbon wasreturned to the shoot from below-ground parts. The total returnof carbon flux to the shoot (85% from nodules) was equivalentto 54% of the total influx of carbon to below-ground parts.Direct carbon transfers between roots and nodules were relativelysmall. Key words: Compartmental analysis, carbon partitioning, root nodules, Glycine max L., ¹³CO₂, assimilation     

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     

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.     

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     

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     

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

From homogenates prepared from surface-sterilized nodules ofseedlings of Casuarina cunninghamiana grown aeroponically, astrain of Frankia designated HFPCc13 was isolated and has beengrown in pure filamentous culture in a defined synthetic nutrientmedium. Vesicle and sporangium formation can be induced by removalof combined nitrogen from the medium.Frankia strain HFPCc13nodulates young seedlings of C. cunninghamiana and C. equisetifoliawithin three weeks of inoculation with an optimum root mediumpH of 6–7 for nodulation and optimum temperature of 30–35^°C. The presence of combined nitrogen in the root mediuminhibits nodulation with NH₄⁺ more inhibitory than NO₃^–.Frankia HFPCc13 does not nodulate Allocasuarina species withinthe same family nor several other possible actinorhizal plantstested. Thus this strain is quite precise in its host specificity.The rate of acetylene reduction was greater in C. cunninghamianathan the closely related species C. equisetifolia. In neitherof these host species were vesicles observed to occur withinthe infected root nodules which had been demonstrated to beactively fixing dinitrogen. Root nodules were shown to be activein acetylene reduction over a range of O₂ concentration in thegaseous environment with an optimum at about 20 per cent O₂,the ambient P_O2 of the air. The mechanism(s) for oxygen protectionof nitrogenase within the filamentous form of Frankia withinthese nodules remains to be explained. Casuarina, Frankia, nodulation, nitrogen fixation     

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.     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 3. Time-course Study on 13C Incorporation into Soluble Metabolites and Respiratory Evolution of 13CO2 from Roots and Nodules

Well-nodulated soya bean (Glycine max L.) plants were allowedto assimilate ¹³CO₂ for 10 h in the light, under steady-stateconditions in which CO₂ concentration and ¹³C abundance wereboth strictly controlled at constant levels. The respiratoryevolution of ¹³CO₂ from roots and nodules and ¹³C incorporationinto various metabolic fractions were measured during the ¹³CO₂feeding and subsequent 48 h chase period. CO₂ respired from nodules was much more rapidly labelled with¹³C than that from roots. The level of labelling (percentageof carbon currently assimilated during the ¹³COM₂ feeding period)of CO₂ respired from nodules reached a maximum of about 87 percent after 4 h of steady-state ^l3CO₂ assimilation and thereafterremained fairly constant. The absolute amount of labelled carbonevolved by the respiration of the nodules during the 10 h ¹³CO₂feeding period was 1·5-fold that of root respiration.These results demonstrated that the currently assimilated (labelled)carbon was preferentially used to support nodule respiration,while root respiration relied considerably on earlier (non-labelled)carbon reserved in the roots. Sucrose pools were mostly composed of currently assimilatedcarbon in all tissues of the plants, since the levels of labellingaccounted for 86–91 per cent at the end of the ¹³CO₂ feeding.In the nodules, the kinetics and levels of sucrose labellingwere in fairly good agreement with those of respired CO₂, whilein the roots, the level of labelling of respired CO₂ was significantlylower than that of sucrose. Succinate and malate were highly labelled in both roots andnodules but they were labelled much more slowly than sucroseand respired CO₂. The kinetics and levels of labelling of theseKrebs cycle intermediates resembled those of major amino acidswhich are derived directly from Krebs cycle intermediates. Itis suggested that large fractions of organic acids in noduleswere physically separate from the respiration site. Glycine max L., Soya bean, ¹³CO₂ assimilation, respiratory evolution of ¹³CO₂, carbon metabolism in root nodules