Acetylene-induced Changes in the Oxygen Diffusion Resistance and Nitrogenase Activity of Legume Root Nodules

Legume symbioses such as pea, lucerne and clover exhibit a substantialdecline in nodular respiration and nitrogenase activity whensubjected to gas streams containing C₂H₂. Assuming a constantrate of O₂ diffusion into the nodule this decrease in respiratoryO₂ consumption would lead to an increase in internal O₂ concentrationwhich would inactivate nitrogenase. No such inactivation hasbeen observed indicating that a change in diffusion resistanceis involved in the C₂H₂ response. Root nodules of C₂H₂ responsivesymbioses are distinguished by their tolerance to high (80 percent) O₂ levels. The nitrogenase of soya-bean and sainfoin,which do not respond to added C₂H₂, is denatured at O₂ levelsabove 40 per cent. Even in O₂-tolerant systems (e.g. pea) theenzyme is damaged if the increase in O₂ levels is rapid (ca.45 s) indicating that an induced change rather than a ‘permanent’feature is involved. The O₂-tolerant, C₂H₂-responsive behaviourpattern of white clover nodules was reversed by pulse feedingwith nitrate-N indicating that the overall C: N ratio in thenodules is involved in determining the response.     

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     

Phloem Glutamine and the Regulation of O2 Diffusion in Legume Nodules

The aim of the present study was to test the hypothesis that the N content or the composition of the phloem sap that supplies nodulated roots may play a role in the feedback regulation of nitrogenase activity by increasing nodule resistance to O2 diffusion. Treating shoots of lupin (Lupinus albus cv Manitoba) or soybean (Glycine max L. Merr. cv Maple Arrow) with 100 [mu]L L-1 NH3 caused a 1.3-fold (lupin) and 2.6-fold (soybean) increase in the total N content of phloem sap without altering its C content. The increase in phloem N was due primarily to a 4.8-fold (lupin) and 10.5-fold (soybean) increase in the concentration of glutamine N. In addition, there was a decline in both the apparent nitrogenase activity and total nitrogenase activity that began within 4 h and reached about 54% of its initial activity within 6 h of the start of the NH3 treatment. However, the potential nitrogenase activity values in the treated plants were not significantly different from those of the control plants. These results provide evidence that changes in the N composition of the phloem sap, particularly the glutamine content, may increase nodule resistance to O2 diffusion and, thereby, down-regulate nodule metabolism and nitrogenase activity by controlling the supply of O2 to the bacteria-infected cells.     

Oxygen Diffusion Pathways and Nitrogen Fixation in Legume Root Nodules

The theoretical analysis presented in this paper suggests thatthe nature of the diffusion pathway from the surface of a noduleto the infected zone depends on the morphology of the nodule;in particular the cross-sectional area of the intercellularspaces in the inner cortex. If the diffusion barrier containscontinuous pathways there will be no pressure difference betweenthe atmosphere and the infected zone. The conditions under whichthe intercellular spaces of the inner cortex could be air-filled,water-filled, or a combination of both are explored. An experimentto resolve this issue is suggested. Information obtained usingcryo-scanning electron microscopy and oxygen electrode datahave been used to illustrate various points. Expressions arederived for the diffusion resistance of the nodule and its componentparts. To provoke further discussion a simple mechanism forthe control of diffusion is described in general terms. Oxygen, diffusion resistance, nodule, nitrogen fixation, soybean, Glycine max. (L.) Merr. cv. Fiskeby V, Trifolium repens L. cv. Blanca, lucerne, Medicago saliva L. cv. Europe     

A Re-Evaluation of the Role of the Infected Cell in the Control of O2 Diffusion in Legume Nodules

Two different simulation models were constructed to describe O2 diffusion into the bacteria-infected cells of legume nodules: one based on a central zone of uniform spherical cells and the other on a central zone of packed, uniform cubical cells with air spaces along the edges. The cubical model more closely approximated the geometry and gas diffusion characteristics of infected cells than did the spherical model. The models relied on set values for the innermost O2 concentration in the infected cell (1-20 nM) and predicted values for the free O2 and oxygenated leghemoglobin gradients toward the cell:space interface. The cubical model but not the spherical model predicted saturation of leghemoglobin (Lb) oxygenation at or within a few micrometers of the gas-filled intercellular space and predicted that the space concentration could be as high as 1.3% O2 when the fractional oxygenation of Lb and respiration rate within the infected cell were typical of that which has been measured in vivo. In the model, the higher the space O2 concentration, the greater the saturation of Lb by O2 and the greater the collapse of Lb-facilitated diffusion near the cell:space interface. This was predicted to result in a greater resistance to O2 diffusion from the space to the bacteroids, thereby providing an intrinsic, homeostatic mechanism for controlling the rate of O2 influx into infected cells. Changes in the physiological features of the simulated cubical infected cell, such as the proportion of the cell as cytosol, the surface area of the cell exposed to a space, the maximum rate of cellular respiration, or the concentration of Lb in the cytoplasm, significantly altered the extent to which the infected cell would be able to regulate its diffusive resistance. These results demonstrate the possibility of a Lb-based mechanism for controlling the O2 concentration within the infected cells. If such a mechanism exists in legume nodules, it would give the infected cell an ability to exercise fine control over its internal environment, a process that could complement a physical diffusion barrier that may exist in the inner cortex or elsewhere in the nodule and provide coarse control over O2 diffusion.     

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

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

Drought Stress,Permeability to O2 Diffusion,and the Respiratory Kinetics of Soybean Root Nodules

In legume nodules, treatments such as detopping or nitrate fertilization inhibit nodule metabolism and N2 fixation by decreasing the nodule's permeability to O2 diffusion, thereby decreasing the infected cell O2 concentration (Oi) and increasing the degree to which nodule metabolism is limited by O2 availability. In the present study we used nodule oximetry to assess and compare the role of O2 limitation in soybean (Glycine max L. Merr) nodules inhibited by either drought or detopping. Compared to detopping, drought caused only minor decreases in Oi, and when the external O2 concentration was increased to raise Oi, the infected cell respiration rate in the drought-stressed plants was not stimulated as much as it was in the nodules of the detopped plants. Unlike those in detopped plants, nodules exposed to moderate drought stress displayed an O2-sufficient respiration rate that was significantly lower than that in control nodules. Despite possible side effects of oximetry in altering nodule metabolism, these results provided direct evidence that, compared to detopping, O2 limitation plays a minor role in the inhibition of nodule metabolism during drought stress and changes in nodule permeability are the effect, not the cause, of a drought-induced inhibition of nodule metabolism and the O2-suffiecient rate of respiration.     

Transport of Nitrate and Calcium into Legume Root Nodules

Nitrate transport into nodulated plants of soybean (Glycinemax), cowpea (Vigna unguiculata) and faba bean (Vicia faba)was investigated. Nitrate entering the root system of soybeandid not pass out of the vascular system into nodular tissuesin detectable quantities. On the other hand, nitrate could passfrom soil through the outer surface of nodules but did not penetratethe infected tissue. Similarly, nitrate was restricted to corticaltissues of cowpea and faba bean. Thus, nitrate cannot inhibitnitrogen fixation as a result of reduction to nitrite by nitratereductase within the bacteroid zone. These results are, however,consistent with an effect of nitrate on an oxygen diffusionresistance located in the nodule cortex. Unlike nitrate, measurable quantities of ⁴⁵calcium were transportedvia the xylem into infected and cortical tissues of soybeannodules: it also passed from the soil into the free space ofthe nodule cortex. Key words: Nitrate, legume nodules, calcium     

The Resistance of the Diffusion Barrier in Nodules of Myrica gale L. Changes in Response to Temperature but Not to Partial Pressure of O2

Rates of C2H2 reduction and CO2 evolution by nodules were measured in a flowthrough system using intact plants of Myrica gale L. Both activities increased linearly with increasing partial pressure of O2 (pO2) up to 18 kPa. The linear relationship between CO2 evolution and pO2 at pO2 values between 6 and 18 kPa suggests that the diffusion barrier has a constant resistance. The lack of a variable resistance was further supported by sustained increases and decreases in nodule activities in response to changes in pO2 in the range of 6 to 20 kPa O2. When pO2 was increased above 20 kPa, C2H2 reduction and CO2 evolution continually declined with time. These results confirm that the diffusion barrier in nodules of M. gale is not variable in response to changes in pO2. The effect of temperature was examined at 8 and 20 kPa O2. Rates of C2H2 reduction and CO2 evolution increased with increasing temperature from 10 to 30[deg]C at both pO2 values. These results indicate that the diffusion resistance of the barrier changes as temperature changes, with the resistance decreasing as temperature increases.     

Oxygen-Induced Membrane Depolarizations in Legume Root Nodules (Possible Evidence for an Osmoelectrical Mechanism Controlling Nodule Gas Permeability)

Various stresses trigger rapid and reversible decreases in the O2 permeability (PO) of legume root nodules. Several possible mechanisms have been proposed, but no supporting data have previously been presented that meet the requirements for both rapidity and reversibility. Stomatal regulation of gas permeability in leaves involves electrically driven fluxes of inorganic osmoticants, so we investigated the possibility of a somewhat similar mechanism in nodules. We used microelectrodes to monitor membrane potential in intact, attached nodules of Glycine max, Medicago sativa, Lotus corniculatus, and Trifolium repens while controlling external O2 concentration and, in the case of G. max, measuring PO with a nodule oximeter. A 1- to 2-min exposure to 100 kPa O2 was found to induce rapid and reversible membrane depolarizations in nodules of each species. This depolarization (which, to our knowledge, is unique to nodules) is accompanied by reversible decreases in PO in G. max nodules. An osmoelectrical mechanism for control of nodule gas permeability, consistent with these data, is presented.     

The Carbon Balance of a Legume and the Functional Economy of its Root Nodules

Budgets for carbon and nitrogen in shoot, root, and nodulesof garden pea (Pisum sativum L.) are drawn up for a 9-d intervalin the life cycle, from data on nitrogen fixation, carbon accumulationin dry matter, respiratory output of plant organs, and organicsolute exchange between shoot and nodulated root. Of the carbon gained photosynthetically by the shoot from theatmosphere 26 per cent is incorporated directly into its drymatter, 32 per cent translocated to the nodules, and 42 percent to the supporting root. Of the nodules’ share, 5per cent is consumed in growth, 12 per cent in respiration,and 15 per cent returned to the shoot via the xylem, as aminocompounds generated in nitrogen fixation. Growth and respirationof the root utilize, respectively, 7 and 35 per cent. The respiratory efficiency of a nodulated root in terms of nitrogenfixation (5.9mg C per mg N₂-N fixed) is found to be very similarto that of an uninoculated root assimilating nitrate (6.2 mgC per mg NO₃-N reduced). The nodules require in growth, respiration,and export 4.1 mg C ( 10.3 mg carbohydrate) for each mg N whichthey fix. The nodules consume 3 ml O₂ for every 1 ml N₂ utilized in fixation. In exporting a milligram of fixed nitrogen the nodules requireat least 0.35 ml of water. Almost half of this requirement mightbe met by mass flow into the nodules via the phloem.     

Diffusion of Gases through Plant Tissues : Entry of Acetylene into Legume Nodules

I have measured acetylene diffusion through plant tissues including nodules from several species of legume—vetch, peas, soybeans, and Sesbania rostrata. The observed half-time for reequilibration of internal and external concentration is less than 1 minute for typical nodules. Inward diffusion of acetylene in air is rapid relative to the use of acetylene by nitrogenase so that diffusion of acetylene would not be a significant limiting factor for nitrogenase activity in air. However, under an atmosphere of Ar:O₂ where there is no N₂ reduction, the inward diffusion rate of acetylene into larger nodules could produce a measurable limitation of observed nitrogenase activity at low acetylene concentrations.     

The Structure of Nitrogen Fixing Root Nodules on the Aquatic Mimosoid Legume Neptunia plena

Nodules of the aquatic mimosoid legume Neptunia plena (L.) Benth.were always found associated with roots but not stems. Theyappeared macroscopically 10 and 20 d after inoculation on plantsgrown hydroponically and in vermiculite, respectively. The developmentof nitrogen-fixing cells occurred in a series of stages notyet reported in legume nodule formation: initial infection wasapparently intercellular and rhizobia spread between cells andthrough intercellular spaces before penetrating individual hostcells by means of infection threads. Subsequently nodule developmentwas broadly similar to that described for indeterminate papilionoidnodules. The infection threads of Neptunia and pea nodules containeda matrix with a common epitope, which was, in Neptunia, extrudedfrom the infection thread at the point of bacterial release. The central tissue contained infected and interstitial cellsand was surrounded by a three-layered cortex and a phellem.Bounding the infected region was a layer two to three cellsthick with large, unoccluded intercellular spaces. Externalto this was a layer, one or more cells thick, in which the cellwalls were interlocked, reducing the number of radially orientedintercellular spaces. The outer layer, several cells thick,contained intercellular spaces many of which were occluded.These features did not vary with growth conditions in a waywhich might influence oxygen diffusion characteristics. However,the phellem of water-cultured nodules was much more aerenchymatousthan that of vermiculite-grown nodules. Aquatic legume, Neptunia plena, nitrogen fixation, oxygen, root nodules, Rhizobium     

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     

Nickel Resistance Determinants in Bradyrhizobium Strains from Nodules of the Endemic New Caledonia Legume Serianthes calycina

Bradyrhizobium strains, isolated in New Caledonia from nodules of the endemic legume Serianthes calycina growing in nickel-rich soils, were able to grow in the presence of 15 mM NiCl₂. The genomes of these strains harbored two Ni resistance determinants, the cnr and nre operons. By constructing a cnrA mutant, we demonstrated that the cnr operon determines the high nickel resistance in Bradyrhizobium strains.     

Malonate Catabolism Does Not Drive N2 Fixation in Legume Nodules

Malonyl-coenzyme A (CoA) decarboxylase, malonyl-CoA synthetase, and malonate transporter mutants of Rhizobium leguminosarum bv. viciae and trifolii fixed N₂ at wild-type rates on pea and clover, respectively. Thus, malonate does not drive N₂ fixation in legume nodules.     

Sucrose Synthase in Legume Nodules Is Essential for Nitrogen Fixation

The role of sucrose synthase (SS) in the fixation of N was examined in the rug4 mutant of pea (Pisum sativum L.) plants in which SS activity was severely reduced. When dependent on nodules for their N supply, the mutant plants were not viable and appeared to be incapable of effective N fixation, although nodule formation was essentially normal. In fact, N and C resources invested in nodules were much greater in mutant plants than in the wild-type (WT) plants. Low SS activity in nodules (present at only 10% of WT levels) resulted in lower amounts of total soluble protein and leghemoglobin and lower activities of several enzymes compared with WT nodules. Alkaline invertase activity was not increased to compensate for reduced SS activity. Leghemoglobin was present at less than 20% of WT values, so O₂ flux may have been compromised. The two components of nitrogenase were present at normal levels in mutant nodules. However, only a trace of nitrogenase activity was detected in intact plants and none was found in isolated bacteroids. The results are discussed in relation to the role of SS in the provision of C substrates for N fixation and in the development of functional nodules.