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 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     

Direct Test for Altered Gas Exchange Rates in Water stressed Soybean Nodules

Water stress usually lowers the nitrogenase activity of soybeanroot nodules. This loss in activity might result from an increasedbarrier to nodular gas exchange, from a general reduction inbiochemical function, or both. To test for the possibility ofan increased barrier to gas diffusion, we measured the apparentlag time for initiation of acetylene reduction by intact soybeanplants, both before and after water stress. Mild nodular waterloss (i.e. 10% of fresh weight or less) seldom altered the apparentlag time, whereas severe water stress (20–40% f. wt loss)frequently produced a small increase in apparent lag time. Severewater stress also produced a large decrease (24%) in the externaldiameter of the nodules and a loss of the white lenticel traces.Water stress usually caused a decrease in the apparent K_m foracetylene. The data do not suggest a large change in the diffusiveresistance to acetylene of nodules subjected to water stress.Thus, the observed decrease in nitrogenase activity may resultprimarily from biochemical, rather than physical, changes. However,because of the relatively greater importance of gas-phase diffusionfor oxygen entry, we cannot exclude the possibility of a largechange in a small gas pathway that affects oxygen influx morethan acetylene influx. Diffusion, Glycine max, nitrogen fixation, water stress     

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     

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     

Relationships between Acetylene Reduction Activity, Hydrogen Evolution and Nitrogen Fixation in Nodules of Acacia spp.: Experimental Background to Assaying Fixation by Acetylene Reduction under Field Conditions

Hansen, A. P., Pate, J. S. and Atkins, C. A. 1987. Relationshipsbetween acetylene reduction activity, hydrogen evolution andnitrogen fixation in nodules of Acacia spp.: Experimental backgroundto assaying fixation by acetylene reduction under field conditions.—J.exp. Bot. 38: 1–12 Glasshouse grown, symbiotically-dependent seedlings of Acaciaalata R.Br., .A. extensa Lindl., and A. pulchella R.Br. wereexamined for acetylene reduction in closed assay systems usingundisturbed potted plants, excavated whole plants, nodulatedroots or detached nodules. Nitrogenase activity declined sharplyover the first hour after exposure of detached nodules to acetylene(10% v/v in air), less steeply or not at all over a 3 h periodin assays involving attached nodules. Using detached nodules,rates of acetylene reduction, nitrogen (¹⁵N₂) fixation, andhydrogen evolution in air (¹⁵N₂) and acetylene-containing atmosphereswere measured in comparable 30 min assays. Total electron flowthrough nitrogenase in air was determined from rates of nitrogen(¹⁵N₂) fixation ( ? 3) plus hydrogen evolution, that in thepresence of acetylene from rates of acetylene reduction andhydrogen evolution in air: acetylene. Values for the ratio ofelectron flow in air: acetylene to that in air ranged from 0?43to 0?83 in A. pulcheila, from 0?44 to 0?66 in A. alala and from0?37 to 0?70 in A. extensa, indicating substantial inhibitionof electron flow through nitrogenase of detached nodules byacetylene. Relative efficiencies of nitrogenase functioningbased on hydrogen evolution and acetylene reduction were from0?15 to 0?79, those based on nitrogen (¹⁵N₂) fixation and hydrogenevolution from 0?53 to 0?87. Molar ratios of acetylene reducedto nitrogen (¹⁵N₂) fixed were 2?82 ? 0?24, 201 ? 0?15, and 1?91? 0?11 (?s.e.; n = 7) for A. pulcheila,A. extensa and A. alata respectively A standard 5–10 min acetylene reduction assay, conductedon freshly detached unwashed nodules in daytime (12.00–14.00h), was calibrated for field use by comparing total N accumulationof seedlings with estimated cumulative acetylene reduction overa 7-week period of glasshouse culture. Molar ratios for acetylenereduced: nitrogen fixed using this arbitrary method were 3?58for A. alata, 4?82 for A. extensa and 1?60 for A. pulchella.The significance of the data is discussed. Key words: Acacia spp, nitrogenase functioning     

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     

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     

Limitations on the Analysis of Acetylene Reduction by Soybean at Low Levels of Acetylene

The analysis of acetylene reduction at low concentrations ofacetylene involves a number of assumptions and both technicaland kinetic complexities. The major difficulty in convertingacetylene reduction rates to apparent N₂ reduction rates isdetermining the K_m for acetylene in the presence of N₂. Thissubstrate competition is dominant over diffusion limitationeffects, but both introduce equivalent deviations in the observedK_m. Because N₂ is a non-linear partial competitive inhibitorof acetylene reduction, correction for its presence is difficult.Two further complications are introduced by the non-linear responseof nitrogenase to acetylene concentration even in the absenceof N₂, and changes in the apparent K_m of acetylene and K₁ ofN₂ as a function of other variables in the enzyme assay. Itis proposed that transient analysis may be used for measurementof diffusion coefficients and calculations of possible diffusionlimitations. It is demonstrated that one proposed model forestimating diffusion limitation (Denison et al., 1983, PlantPhysiology 73, 648–51) confounds substrate competitionwith diffusion limitation. Acetylene reduction, nitrogen fixation, diffusion limitation     

Inhibition of nitrogenase activity and nodule oxygen permeability by water deficit

Short-term effects of water deficit on nitrogenase activitywere investigated with hydroponically grown soybean plants (Glycinemax L. Merr. cv. Biloxi) by adding polyethylene glycol (PEG)to the hydroponic solution and measuring nitrogenase activity,nodule respiration, and permeability to oxygen diffusion (P_o).These experiments showed a rapid decrease in acetylene reductionactivity (ARA) and nodule respiration. A consequence of thedecreased respiration rate was that P_o calculated by Fick'sLaw also decreased. However, these results following PEG treatmentwere in direct conflict with a previous report of stabilityin P_o determined by using an alternative technique. To resolvethis conflict, an hypothesis describing a sequence of responsesto the initial PEG treatment is presented. An important findingof this study was that the response to water deficit inducedby PEG occurred in two stages. The first stage of decreasednodule activity was O₂-limited and could be reversed by exposingthe nodules to elevated pO₂. The second stage which developedafter 24 h of exposure to PEG resulted in substantial loss innodule activity and this activity could not be recovered withincreased pO₂. Severe water deficit treatments disrupt noduleactivity to such a degree that O₂ is no longer the major limitation. Key words: Glycine max, N₂ fixation, soybean, oxygen permeability, water deficit     

Alterations in Apoplastic and Total Solute Concentrations in Soybean Nodules Resulting from Treatments Known to Affect Gas Diffusion

Ureide concentration in the cortical apoplast of soybean (Glycinemax(L.) Merr.) nodules increases rapidly in response to noduleexcision. The objective here was to determine if changes inapoplastic ureide may be related to the control of resistanceto gas diffusion which is thought to be localized in the nodulecortex. Following decapitation of shoots, nitrogenase activity(acetylene reduction) and ureide concentration in total noduleextracts declined over a period of several hours. Apoplasticureide concentration relative to total nodule ureide was elevatedunder these conditions, but the treatment effect was small comparedto non-decapitated controls. Decapitation also caused a significantdecline in the concentrations of sucrose, glucose, and D-pinitolin nodules. However, the decline in carbohydrates was similarin the nodule cortex and the nodule as a whole, suggesting thatthe carbohydrate changes are not related to a cortex-localizedmechanism. Non-invasive treatments involving increases or decreasesin oxygen concentration supplied to nodulated roots caused rapiddecreases in respiration of nodulated roots and in ureide concentrationin total nodule extracts, but did not cause major changes inapoplastic ureide concentrations. The combined results indicatethat apoplastic ureide is probably not involved in the regulationof resistance to gas diffusion. The rapid decline in noduleureide concentrations in response to changing oxygen supplydocuments the sensitivity of ureide synthesis and/or transportto alterations in nodule respiration and/or nitrogenase activity Key words: Glycine max, Pisum sativum, ureide, carbohydrates     

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     

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     

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     

Seedling Nitrate Reductase Activity and Nitrate Partitioning in Maize (Zea mays L.) Strains Divergently Selected For Post-anthesis Leaf-Lamina Nitrate Reductase Activity

Two experiments were conducted to evaluate the effects of phenotypicrecurrent selection for high and low post-anthesis leaf-laminain vivo NRA on nitrate uptake, nitrate partitioning and in vitroNRA of seedling roots and leaves. In Experiment 1, intact plantsof cycle 0, 4, and 6 of the high and low NRA strains were grownon NH₄-N for 11 d, then exposed to 1.0 mol m^–3 KNO₃, andcultures sampled at 6 h and 28 h (induction and post-inductionperiods). Nitrate uptake, tissue nitrate concentration and invitro NRA were determined. The pattern of response to selectionin seedling leaf NRA was similar to that observed for in vivoNRA of field grown plants. Leaf NRA increased between 6 h and28 h. Root NRA was not affected by selection or sampling time.Treatments differed in total fresh weight but not in reductionor uptake of nitrate per unit weight, indicating a lack of correspondencebetween NRA and reduction and supporting the idea that concomitantreduction by NR is not obligatorily linked to nitrate influxin the intact plant. In Experiment 2, dark-grown plants of cycle 0, and 6 of thehigh and low NRA strains were cultured without N, detopped onday 6, transferred the following day to 0-75 mol m^–3 KNO₃and sampled at 6 h and 28 h. In contrast to Experiment 1, selectionpopulations differed in nitrate reduction and root NRA, whichby 28 h reached higher average levels than root NRA of intactplants. Translocation and reduction were inversely related amongstrains within each sampling time. The high level of translocationin detopped plants of the low NRA strain was difficult to reconcilewith its low leaf NRA level of Experiment 1. It is suggestedthat nitrate transport in detopped roots is altered relativeto the intact system in a way which permits greater NRA inductionand nitrate reduction. The results indicate that nitrate partitioningby detopped root systems should be interpreted with caution. Key words: Zea, nitrate reductase activity, nitrate uptake, nitrate reduction, nitrate partitioning, selection     

Nitrogenase Activity, Photosynthesis and Nodule Water Potential in Soyabean Plants Experiencing Water Deprivation

The effects of water deprivation on the activity of nodulesof soyabean (Glycine max) have been investigated in controlledenvironments. The water potential of detached nodules was measuredusing a Wescor psychrometer and a specially designed ten chamberpsychrometer; each chamber was capable of holding up to sixnodules which allowed for simultaneous measurements of betweenten and 60 nodules. Nitrogenase activity (acetylene reduction)and respiration of intact, undisturbed nodulated roots was measuredin a flow-through gas system. The equilibration pattern of singlenodules in a Wescor chamber showed a distinct biphasic pattern.Differences between the pseudo- and the true equilibrium valuesof water potential suggest that water potential gradients closeto 01 MPa exist within the nodule tissue. Such gradients probablyreflect histological discontinuities. When detached noduleswere allowed to dry, decreases in water potential and diameterwere accompanied by increases in the resistance to water vapourloss through diffusion. These changes provide evidence for anatomicalmodifications which might provide some regulation of water loss. During a 7 d period of water deprivation there was a close relationshipbetween decreases in leaf and nodule water potential. Nitrogenaseactivity showed a 70% decrease during the first 4 d, whilstphotosynthesis only declined by 5%. It is suggested that waterstress exerts an influence on nitrogenase activity which isindependent of the rate of photosynthesis; it acts directlyon nodule activity through increases in the resistance to oxygendiffusion to the bacteroids. The data suggest that the linearrelationship between oxygen diffusion resistance and water potentialis more important than any reductions in photosynthate supply. Key words: Nitrogen fixation, water potential, photosynthesis     

RNA Synthesis in Phaseolus Chloroplasts: II. RIBONUCLEIC ACID SYNTHESIS IN CHLOROPLASTS FROM DEVELOPING AND SENESCING LEAVES

The rate of RNA synthesis in chloroplasts from the primary leavesof Phaseolus vulgaris L. cv. Canadian Wonder was measured invitro as plant age increased. The rate per leaf began to fallbefore the leaf was 70% expanded. At full expansion, activityhad fallen by 70%. Chloroplast RNA synthesis per unit chlorophyllwas falling before the leaf was 25% expanded. When all parts of the plant above the mature primary leaveswere removed (detopping) chloroplast RNA synthesis in theseleaves rose within 36 h. The rate increased to a maximum 3–4d after detopping, when it was 5–10 times control values;thereafter it fell again. The chlorophyll content began to increaseabout 4 d after detopping, eventually rising by 100%. Detoppingcaused a 3-fold increase in the Triton X-100-soluble DNA contentof chloroplast preparations, measured after 3.5 d. At that timethe rate of RNA synthesis per unit Triton-soluble DNA was thesame in chloroplasts from the primary leaves of intact and detoppedplants. Detopping also resulted in an increase in the depthof the leaf palisade layer. The effects of detopping on chloroplasts were prevented by darknessand reduced by shading. Increased chloroplast RNA polymerase activity was also inducedin the primary leaves by placing a polythene bag over intactplants, enclosing everything above these leaves. Removal ofthe roots from detopped plants prevented the rise in the rateof chloroplast RNA synthesis.     

Nitrogenase (C2H2) Activities of Isolated Peanut and Cowpea Bacteroids at Optimal Oxygen Availability and Comparison with Whole Nodule Activities

Bacteroids, formed by the same strain of Rhizobium, were isolatedanaerobically from peanut and cowpea root nodules and theirC₂H₂ reduction activities were measured. Measurements were startedin a pure N₂ atmosphere followed by stepwise addition of smallamounts of O₂. The procedures may have general application andare described in detail. With increasing O₂ level, a gradualincrease in nitrogenase activity was observed which reacheda peak, presumably at the optimum availability of O₂ to bacteroids,and then declined. The maximum activity attained by isolatedbacteroids of cowpea was much higher than that obtained frommeasurement of activities of intact nodules and their bacteroidcontent, whereas for peanut the two were nearly equal. The resultsindicated that intranodular conditions are probably responsiblefor the difference in nitrogenase activities of peanut and cowpeanodules rather than the unique morphological modification ofpeanut bacteroids. Key words: Root nodules, Peanut, Cowpea, Bacteroids, Nitrogenase activity     

Oxygen Diffusion in Lupin Nodules: I.VISUALIZATION OF DIFFUSION BARRIER OPERATION

Root nodules of Lupinus albus (L.) cv. Multolupa were subjectedto short- and medium-term stresses by lowering rhizosphere temperaturefrom 25 to 16°C (2 h), detopping plants (3 h), darkeningplants (21 h) or exposing roots to 20 mol m^–3 KNO₃ for4 d. All experimental treatments produced increases in oxygendiffusion resistance, compared with control plants. These correlatedwith structural changes in the nodule cortex, which is describedin detail for the first time. The most noticeable change isthe occlusion of intercellular spaces by a glycoprotein whichwas identified using the monoclonal antibody MAC236. This glycoproteinwas also found surrounding bacteria in intercellular spacesof the cortex of control nodules. Key words: Oxygen diffusion resistance, glycoprotein, nodules, nitrogen fixation, Lupinus albus     

Metabolic changes in soybean nodules after inhibition of nitrogen fixation by treatment with oxygen

Metabolites that accumulated in soybean [Glycine max (L.) Merr.]nodules after inhibition of nitrogen fixation were analysedto determine what carbon compounds the bacteroids might obtainfrom their host. Exposure of roots of intact soybean plantsto 100% O₂ for 5 min caused a decrease in acetylene reductionactivity within 10 min and then the activity recovered onlyslowly. Analysis of carbohydrates, organic acids, volatile compoundsand amino acids in extracts of nodules revealed that succinate,malate and alanine all accumulated within 10 min after treatmentwith O₂. The concentrations of sucrose, acetone, tyrosine, valine,isoleucine, leucine, and ornithine in the nodules increasedslowly after such treatment. The results are discussed in termsof carbon sources for supporting nitrogen fixation of soybeanbacteroids. Key words: Glycine max, carbon metabolism, nitrogen fixation, nodules