Delivery of N2 to bacteroids in simulated soybean nodule cells: consideration of gradients of concentration of dissolved N2 in cell walls,cytoplasm, and symbiosomes

Summary The previously published simulation of physiological functions occurring in infected cells of soybean nodules has been extended to include consideration of the diffusion of N₂ from the outside of a nodule to the nitrogen-fixing bacteroids, in relation to published values for the apparentK _m(N₂) for nitrogen fixation in the soybean nodule system. Nitrogen fixation is driven by bacteroid respiration, so increases in the average relative oxygenation (Y) of cytoplasmic leghaemoglobin lead to increased bacteroid respiration, increased nitrogen fixation, and greater differences in concentration of dissolved N₂ between the cell surface and the innermost bacteroids (d[N₂]). Over the range ofY considered, values for d[N₂] were from 5.2- to 6.2-fold greater than the corresponding values for d[O₂], because of facilitation of O₂ flux by cytoplasmic leghaemoglobin. Gradients of [N₂] within symbiosomes are small relative to cytoplasmic values and at the symbiosome surface [N₂] was greater than 0.4 mol/m³ at the greatest rates of nitrogen fixation calculated. Therefore, it is unlikely that values for [N₂] anywhere in the infected cell are low enough to affect rates of nitrogen fixation significantly, unless low external atmospheric N₂ pressures are used experimentally.Abbreviations Lb leghaemoglobin - LbO₂ oxyleghaemoglobin - [O₂], [N₂ concentrations of free, dissolved oxygen and nitrogen - Y fractional oxygenation of leghaemoglobin     

Diffusion Limitation of Oxygen Uptake and Nitrogenase Activity in the Root Nodules of Parasponia rigida Merr. and Perry

Parasponia is the first non-legume genus proven to form nitrogen-fixing root nodules induced by rhizobia. Infiltration with India ink demonstrated that intercellular air spaces are lacking in the inner layers of the nodule cortex. Oxygen must diffuse through these layers to reach the cells containing the rhizobia, and it was calculated that most of the gradient in O₂ partial pressure between the atmosphere and rhizobia occurs at the inner cortex. This was confirmed by O₂ microelectrode measurements which showed that the O₂ partial pressure was much lower in the zone of infected cells than in the cortex. Measurements of nitrogenase activity and O₂ uptake as a function of temperature and partial pressure of O₂ were consistent with diffusion limitation of O₂ uptake by the inner cortex. In spite of the presumed absence of leghemoglobin in nodules of Parasponia rigida Merr. and Perry, energy usage for nitrogen fixation was similar to that in legume nodules. The results demonstrate that O₂ regulation in legume and Parasponia nodules is very similar and differs from O₂ regulation in actionorhizal nodules.     

Lignification of cell walls of infected cells in Casuarina glauca nodules that depend on symplastic sugar supply is accompanied by reduction of plasmodesmata number and narrowing of plasmodesmata

The oxygen protection system for the bacterial nitrogen‐fixing enzyme complex nitrogenase in actinorhizal nodules of Casuarina glauca resembles that of legume nodules: infected cells contain large amounts of the oxygen‐binding protein hemoglobin and are surrounded by an oxygen diffusion barrier. However, while in legume nodules infected cells are located in the central tissue, actinorhizal nodules are composed of modified lateral roots with infected cells in the expanded cortex. Since an oxygen diffusion barrier around the entire cortex would also block oxygen access to the central vascular system where it is required to provide energy for transport processes, here each individual infected cell is surrounded with an oxygen diffusion barrier. In order to assess the effect of these oxygen diffusion barriers on oxygen supply for energy production for transport processes, apoplastic and symplastic sugar transport pathways in C. glauca nodules were examined. The results support the idea that sugar transport to and within the nodule cortex relies to a large extent on the less energy‐demanding symplastic mechanism. This is in line with the assumption that oxygen access to the nodule vascular system is substantially restricted. In spite of this dependence on symplastic transport processes to supply sugars to infected cells, plasmodesmal connections between infected cells, and to a lesser degree with uninfected cells, were reduced during the differentiation of infected cells.     

Gas diffusion pathway in nodules ofCasuarina cunninghamiana

The gas diffusion pathway in nodules was traced by vacuum infiltration with India ink or aniline blue and by electron microscopy. India ink infiltration was observed in the outermost and the innermost cortex in sliced nodules, but not in intact nodules. With aniline blue infiltration, it was observed that intercellular air spaces in the outermost and the innermost cortex were connected to those in nodule roots. No air spaces were in contact with walls of infected cells, although intercellular air spaces existed in some groups of uninfected cells within the infected zone. Infiltration with either India ink or aniline blue could not be observed in the infected zone in essentially all cases. Thus it is suggested that the discontinuity of the intercellular air spaces represents a major resistance to O₂ diffusion in nodules ofCasuarina cunninghamiana.     

A Model of the Regulation of Nitrogenase Electron Allocation in Legume Nodules (I. The Diffusion Barrier and H2 Inhibition of N2 Fixation)

A mathematical model is presented to explain the regulation of nitrogenase electron allocation to N2 fixation (EAC) in legume nodules. The model is based on two assumptions: (a) that H2 inhibits N2 fixation in a competitive manner; and (b) that O2, H2, and N2 move into and out of nodules by diffusion and their movement is impeded by a diffusion barrier, the permeability of which is controlled to maintain a very low infected cell O2 concentration. When the model was used to simulate nodules displaying a range of values for total nitrogenase activity (TNA), maximum EAC values were predicted to be between 0.69 and 0.71, and a negative correlation was predicted to exist between EAC and TNA. These predictions were in good agreement with empirically derived values reported in the literature and support the suggestion that H2 inhibition of N2 fixation is a major determinant in the regulation of nitrogenase EAC in legume nodules. Two versions of the model were constructed. A closed-pore model assumed that the diffusion barrier consisted of a solid shell of water of variable thickness in the nodule cortex. An open-pore model assumed that a small number of gas-filled intercellular spaces connected the nodule central zone with the root atmosphere and these pores were opened or closed by water to provide variations in the nodule's permeability to gas diffusion. Because of differences in the diffusivity of gases in the gaseous and aqueous phases, the model predicted that, at a given infected cell O2 concentration, an open-pore diffusion barrier would result in less H2 accumulation in the infected cells than a closed-pore diffusion barrier. Therefore, the model may be used to test specific hypotheses about the physical structure of the barrier to gas diffusion in legume nodules.     

Mathematical modeling of oxygen diffusion and respiration in legume root nodules

The O₂ permeability of legume root nodules is under physiological control; decreases in permeability are triggered by various forms of stress. Two linked mathematical models were used to explore several hypotheses concerning the physical nature of the variable diffusion barrier in nodules. Respiration and diffusion of dissolved O₂ and oxygenated leghemoglobin were simulated for the nodule cortex and the nodule interior. Measured nodule permeabilities were shown to be inconsistent with the hypothesis that large numbers of air-filled pores penetrate the diffusion barrier. Changes in the affinity of leghemoglobin for O₂ or in the rate of cytoplasmic streaming in diffusion barrier cells did not result in the large changes in O₂ permeability reported for real nodules. The presence or absence, but not the thickness, of aqueous plugs in radial pores through the cortex was found to have a large effect on permeability. Flooding of intercellular spaces, either between layers of cells in the cortex or in the nodule interior, also caused large changes in simulated permeability. The unsteady-state O₂ method for determining nodule permeability was tested using data generated by the model. The accuracy of the method was confirmed, provided that certain assumptions (full oxygenation of leghemoglobin under pure O₂ and uniform conditions in the nodule interior) are met.     

The occurrence of leghemoglobin protein in the uninfected interstitial cells of soybean root nodules

The distribution of leghemoglobin (Lb) in resin-embedded root nodules of soybean (Glycine max (L.) Merr.) was investigated using immunogold labeling. Using anti-Lb immunoglobulin G and protein A-gold, Lb or its apoprotein was detected both in cells infected by Bradyrhizobium japonicum and in uninfected interstitial cells. Leghemoglobin was present in the cytoplasm, exclusive of the organelles, and in the nuclei of both cell types. In a comparison of the density of labeling in adjacent pairs of infected and uninfected cells, Lb was found to be about four times more concentrated in infected cells. This is the first report of Lb in uninfected cells of any legume nodule; it raises the possibility that this important nodule-specific protein may participate in mediating oxygen flow to host plant organelles throughout the infected region of the nodule.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - kDA kilodalton - Lb leghemoglobin - TBST Tris-buffered saline plus Tween 20     

Tissue distribution and subcellular localization of carbonic anhydrase in mature soybean root nodules indicates a role in CO2 diffusion

Tissue distribution of carbonic anhydrase (CA; EC 4.2.1.1) and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) was examined in developing soybean (Glycine max) nodules using an immunohistological approach. The data obtained indicate that in young nodules both CA and PEPC proteins are present in the parenchymatous cells, and at much lower levels, in the central nodular region. In mature nodules, high levels of CA were exclusively present in 2-3 cell layers of the inner cortical region, whereas high levels of PEPC were present both in infected and uninfected cells. Immunogold localization indicated that, in mature nodules, CA was localized in the cytoplasm of the inner cortical cells and the cell walls of the endodermal cells. These results considered together suggest that in mature nodules, CA may facilitate the diffusion of the excessive CO₂, derived from the respired bacteroids, in the rhizosphere. The distribution of CA was examined in mature nodules of soybean, grown hydroponically, in either limiting or non-limiting phosphate concentrations. The data indicated that in plants growing on non-limiting phosphate concentrations, an additional strong signal was found in cortical cells surrounding the nodule vascular bundles.     

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.     

Distribution of O2 within infected cells of soybean root nodules: a new simulation

Summary A simulation model is presented for the distribution and consumption of O₂ in infected cells of soybean root nodule central tissue. It differs from earlier models in closer adherence to observed structure and embodies new morphometric data about the distribution of > 12,000 mitochondria per cell and about the geometry of the gas-filled intercellular spaces near which the mitochondria are located. The model cell is a rhombic dodecahedron and O₂ enters only through interfaces (totalling 26% of the cell surface) with 24 gas-filled intercellular spaces. These spaces are located at the edges of each rhombic face of the cell, forming an interconnected network over the cell suface. Next, O₂ is distributed through the cytoplasm by a leghaemoglobin-facilitated diffusive process, initially between the mitochondria and amyloplasts in the outer layers of the cell and then between > 6,000 symbiosomes (each containing 6 bacteroids) towards the central nucleus. The symbiosomes and mitochondria consume O₂, but impede its diffusion; all O₂ entering symbiosomes is considered to be consumed there. For the calculations, the cell is considered to consist of 24 structural units, each beneath one of the intercellular spaces, and each is divided into 126 layers, 0.2 m thick, in and through which O₂ is consumed and diffused. Rates of consumption of O₂ and of N₂ fixation in each diffusion layer were calculated from previously-established kinetics of respiration by mitochondria and bacteroids isolated from soybean nodules and from established relationships between bacteroid respiration and N₂ fixation. The effects of varying the O₂-supply concentration and the concentration and type of energy-yielding substrates were included in the simulations. When the model cell was supplied with 0.5 mM malate, mitochondria accounted for a minimum of 50% of the respiration of the model cell and this percentage increased with increased concentration of the O₂ supply. Gradients of concentrations of free O₂ dissolved in the cytoplasm were steepest near the cell surface and in this location respiration by mitochondria appeared to exert a marked protective effect for nitrogen fixation in layers deeper within the cell. Estimates of N₂ fixation per nodule, calculated from the model cell, were similar to those calculated from field measurements.Abbreviations Lb leghaemoglobin - LbO₂ oxyleghaemoglobin - [O₂] concentration of free, dissolved O₂ - e.m. electron micrograph Dedicated to the memory of Professor John G. Torrey     

3-Phosphoglycerate dehydrogenase in Mesorhizobioum loti is essential for maintaining symbiotic nitrogen fixation of Lotus japonicus root nodules

Mesorhizobium loti is a Gram negative bacterium that induces N₂-fixing root nodules on the model legume Lotus japonicus. Proteomic analysis in M. loti indicated that 3-phosphoglycerate dehydrogenase (EC. 1.1.1.95, PHGDH) protein content was 2.2 times higher in bacteroids than in cultured bacteria. A M. loti mutant (STM5) with a transposon insertion in the PHGDH gene, mll3875, showed an absolute dependence on serine or glycine in minimal medium for growth. When L. japonicus plants were infected with STM5, the roots formed nodules in numbers comparable to those formed by wild type M. loti; however, the nodules showed very low acetylene reduction activity, and significant starch granule accumulation was observed in the uninfected cells. In such nodules, vast necrosis occurred in the central tissue of the nodules, although bacteroids were detected in the infected cell of the nodules. These data indicate that serine or glycine biosynthesis by PHGDH is important for maintaining symbiosis and nitrogen fixation in L. japonicus nodules.     

Photosystem II and oxygen regulation in Sesbania rostrata stem nodules

The tropical wetland legume Sesbania rostrata Brem. produces nitrogen-fixing stem nodules which are green and contain chlorophyll, the chloroplasts being concentrated in a hand in the inner and mid-cortex close to the nitrogen-fixing cells. The photosystem II thylakoid membrane proteins D₁, D₂ and PsbO, which are essential for photo-synthetic O₂ evolution, were shown by immunoblotting to be present in extracts of leaves and stem nodules. Immunogold labelling confirmed their presence on stem nodule thylakoids and showed that labelling was most intense in well-developed chloroplasts in the mid-cortex and least intense in the smaller, less-abundant chloroplasts adjacent to the nitrogen-fixing cells. Concentrations of the oxygen-carrying protein leghaemoglobin (Lb) did not differ between stem and S. rostrata root nodules, and Lb was localized in bacteroid-containing cells, including those immediately adjacent to the cortex, in both nodule types. Moreover, nitrogenase component 2 was localized in bacteroids within the outermost layers of infected cells, suggesting that a low pO₂ was maintained, despite the nearby chloroplasts. Nodule extracts examined by ELISA and immunoblots, using the monoclonal antibody MAC265, showed greatly enhanced expression of a 139 kDa glycoprotein in stem compared to root nodules. Immunogold labelling showed that material containing the MAC265 antigen occluded intercellular spaces, and was present in cell walls, throughout the cortex of stem nodules (particularly in the chloroplasl-rich inner and mid-cortex), but was considerably less evident in root nodules.     

Evidence that short-term regulation of nodule permeability does not occur in the inner cortex

Regulation of nodule permeability in response to short-term changes in environmental and physiological conditions is thought to occur by occlusion of intercellular spaces in the nodule inner cortex. To test this hypothesis, the permeability of legume nodules was altered by adapting them to either 20 or 80% O₂ over a 2.5-h period. The nodules were then rapidly frozen, cryo-planed and examined under cryo-scanning electron microscopy for differences in the number, area or shape factor of intercellular spaces. Comparisons were made between whole nodules and specific nodule zones (outer cortex, middle cortex, inner cortex and central zone) in each treatment. Gas analysis measurements indicated that nodules equilibrated at 20% O₂ had a 6.6-fold higher permeability than those equilibrated at 80% O₂ However, no significant differences were observed between pO₂ treatments in the number of open intercellular spaces, the cross-sectional area of those spaces, or the proportion of the tissue area present as open space in whole nodules or any nodule zone. Also, although nodules in both treatments possessed a boundary layer of tightly packed cells in the inner cortex, the total area of intercellular spaces between cells bordering this layer did not differ between treatments. Together these observations do not support the currently favored hypothesis that nodule permeability is regulated by opening or occlusion of intercellular spaces in the nodule inner cortex. Highly significant differences (P= 0.0006) were observed between O₂ treatments in the shape factor of the open intercellular spaces in all nodule zones. Nodules equilibrated at 80% O₂ had significantly more isodiametric spaces while those equilibrated at 20% O₂ had more long, narrow spaces. This observation suggests that the critical step in the regulation of nodule permeability to O₂ may be localized in the central, infected zone and involve changes in the ratio of the surface area of the intercellular space to the volume of the infected cell.     

Regulation of nitrogen fixation in infected cells of leguminous root nodules in relation to O2 supply

Respiration and nitrogen fixation in legume root nodules is considered to be limited by the rate at which O₂ from the atmosphere can enter nodules. A thin diffusion barrier in the inner cortex, restricts access to the central tissue where there is a high demand for and low concentration of O₂. Observed variations in rates of nodule activities in response to imposed stresses, are often attributed to variations in the diffusion resistance of the barrier. In the present work, alternative or supplementary metabolic mechanisms are considered. Aspects of nodule structure and of metabolism underlying nodule activities are reviewed in terms of components of the symbiotic system, the nature of steady states and in relation to homeostasis of low concentration of O₂ within the bacteroid-filled host cells. It is suggested that variations in O₂-demand of both mitochondria and bacteroids, serve to preserve nitrogenase activity by poising O₂ concentration within safe limits. Further, data from isolated soybean bacteroids suggest that nitrogenase is converted to a less active but more robust form, in the presence of O₂ in excess of about 70 nM, thus protecting nitrogenase from irreversible inactivation by excess O₂. This regulation is rapidly-reversible when O₂ concentration falls below about 0.1 µM. Respiration by large numbers of host mitochondria in the periphery of infected nodule cells, adjacent to gas-filled intercellular spaces, is considered to play an important part in maintaining a steep gradient of O₂ concentration in this zone. Also, it is possible that variations in nodule O₂ demand may be involved in the apparent variations in resistance of the diffusion barrier. It is concluded that there are many biochemical components which should be considered, along with possible changes to the diffusion barrier, when the effects of imposed stresses on nodule activities are being analysed.     

Adenylate gradients and Ar:O(2) effects on legume nodules: I. Mathematical models

Mathematical models were developed to test the likelihood that large cytosolic adenylate concentration gradients exist across the bacteria-infected cells of legume nodules. Previous studies hypothesized that this may be the case to account for the unusually low adenylate energy charge (AEC; 0.65) measured in the plant fraction of metabolically active nodules (M.M. Kuzma, H. Winter, P. Storer, I. Oresnik, C.A. Atkins, D.B. Layzell [1999] Plant Physiol 119: 399-407). Simulations coupled leghemoglobin-facilitated O(2) diffusion into the infected cell, through bacteroid nitrogenase activity, with the ATP demand for transport and ammonia assimilation in the plant fraction of ureide- and amide-producing nodules. Although large cytosolic adenylate gradients were predicted to exist in both nodule types, amide nodules were predicted to have steeper AEC gradients (0.82-0.52) than ureide nodules (0.82-0.61). The differences were attributed to an additional ATP demand for Asn synthesis in the amide nodule. Simulations for nodules transferred to an Ar:O(2) atmosphere predicted a major reduction in the magnitude of adenylate gradients and an increase in the AEC of the plant fraction. Results were consistent with a number of experimental studies and were used to propose an experimental test of the models.     

Mechanism of Nitrogenase Inhibition in Soybean Nodules : Pulse-Modulated Spectroscopy Indicates that Nitrogenase Activity Is Limited by O(2)

A novel, pulse-modulated spectroscopic system for measuring fractional leghemoglobin oxygenation and infected cell O₂ concentration (O_i) in intact attached nodules of soybean (Glycine max) is described. The system is noninvasive and uses a pulsed (1000 Hertz) light-emitting diode coupled to an optical fiber to illuminate the nodule with light at 660 nanometer. A second optical fiber receives a portion of the light reflected from the nodule and directs this to a photodiode. A lock-in amplifier measures only the signal from the photodiode which is in phase with the pulsed light from the light-emitting diode, and the voltage output from the amplifier, proportional to reflectance, is used to calculate fractional leghemoglobin oxygenation and the nanomolar concentration of free O₂ in the infected cells of the nodule (O_i). The system was used to show that inhibition of nitrogenase activity in soybean nodules by NO₃⁻ treatment, stem-girdling, continuous darkness, or nodule disturbance is caused by a reduction in O_i and limitation of respiration in support of nitrogenase activity. A plot of nitrogenase activity (measured as peak H₂ evolution in Ar:O₂) versus O_i for the various treatments was consistent with the concept that O_i limits in vivo nitrogenase activity in legume nodules under adverse conditions. The potential for using O_i to estimate nitrogenase activity in laboratory and field-grown legumes is discussed.     

A new method to detect the presence of continuous gas-filled pathways for oxygen diffusion in legume nodules

A new method is presented which evaluates the contribution ofgas-filled and water-filled pathways across the barrier whichrestricts O₂ diffusion into infected cells of nodules. SinceO₂ will move through air about 104 times faster than throughwater any continuous gas-filled pathways which traverse thecortex would form a major route for O₂ transport. However, microscopicevidence for the existence of such direct connections is ambiguous. On theoretical grounds, O₂ should diffuse through a He atmosphereabout 3.7 times faster than through air or Ar, but O₂ flux acrossa liquid barrier should be unaffected by changes in the backgroundmixing gas. Thus, if O₂ influx to the infected cells is increasedwhen the ambient gas phase is changed from air to He/21% O₂this is evidence for a continuous pathway of gas-filled poresacross the whole width of the cortical barrier. This theoreticalapproach was validated by measuring the rates of diffusion ofO₂ through milli-pore filters in background atmospheres of eitherair, Ar or He. These membranes were used dry, to simulate the‘open pore’ model for nodule diffusion, or wettedwith water or gum Arabic (which is similar to the glycoproteinsassociated with the barrier) to simulate the ‘closed pore’situation. Knowing the diffusion constants for the gas- and water-filledpathways that may be involved in the nodule barrier, we thenevaluated the contribution of these components to the totaldiffusion resistance by examining the effect of Ar/O₂ and He/O₂gas mixtures on H₂ production and respiration of nodules. Theresults indicate that, in unstressed soyabean nodules, abouthalf of the O₂ flux to infected cells is via inter-connectedgas-filled pores, which ‘close’ to produce a liquid-filledbarrier as the diffusion resistance increases in response tostress. In unstressed lupin nodules all of the O₂ flux crossesa liquid-filled barrier. Key words: Oxygen diffusion, nodules, nitrogen fixation     

Spatial relationships between uninfected and infected cells in root nodules of soybean

In soybean (Glycine max (L.) Merr.) the uninfected cells of the root nodule are responsible for the final steps in ureide production from recently fixed nitrogen. Stereological methods and an original quantitative method were used to investigate the organization of these cells and their spatial relationships to infected cells in the central region of nodules of soybean inoculated with Rhizobium japonicum strain USDA 3I1B110 and grown with and without nitrogen (as nitrate) in the nutrient medium. The volume occupied by the uninfected tissue was 21% of the total volume of the central infected region for nodules of plants grown without nitrate, and 31% for nodules of plants grown with nitrate. Despite their low relative volume, the uninfected cells outnumbered the much larger infected cells in nodules of plants grown both without and with nitrate. The surface density of the interface between the ininfected and infected tissue in the infected region was similar for nodules in both cases also, the total range being from 24 to 26 mm²/mm³. In nodules of plants grown without nitrate, all sampled infected cells were found to be in contact with at least one uninfected cell. The study demonstrates that although the uninfected tissue in soybean nodules occupies a relatively small volume, it is organized so as to produce a large surface area for interaction with the infected tissue.     

Delivery of O2 to bacteroids in soybean nodule cells: Consideration of gradients of concentration of free,dissolved O2 in and near symbiosomes and beneath intercellular spaces

Summary Based on a simulation model of the structure of and distribution of O₂ within infected cells of soybean nodules, gradients of concentration of dissolved O₂ ([O₂]) have been calculated within and between symbiosomes embedded in host cytoplasm, through which the flux of O₂ to the symbiosomes is facilitated by leghaemoglobin. As a consequence of facilitation, gradients of [O₂] in cytoplasm between symbiosomes are very small. Within symbiosomes, from which leghaemoglobin is considered to be absent, respiration by bacteroids generates steeper gradients of [O₂], thus restricting respiration and N₂ fixation. However, if bacteroid mass is considered to be randomly distributed within a symbiosome, about 80% of this mass lies within about 0.6 m of the surface (the peribacteroid membrane). Consequently, respiration within a symbiosome was calculated to be between 65% and 92% of that attained if bacteroids were directly in contact with the cytoplasm. For N₂ fixation, the corresponding values were 44% to 91%. In cytoplasm, near the surface of a symbiosome, there is a boundary layer in which equilibrium between O₂, leghaemoglobin and oxyleghaemoglobin is perturbed by O₂ consumption within. Calculations of the thickness of the boundary layers gave values of only 3.65 to 3.75×10^–9 m, thus they had little effect on calculated gradients of [O₂] in cytoplasm. In contrast, perturbations of the leghaemoglobin oxygenation equilibrium affected layers of cytoplasm beneath intercellular spaces to a depth of 0.15 to 0.3×10^–6 m in the physiological range of volume average [O₂]. This affected gradients of [O₂] in the cytoplasm near intercellular spaces. Revisions have been made to the model cell, incorporating these new calculations. Results suggest that infected nodule cells may be able to withstand 1–2 M O₂ in the outermost layers of cytoplasm without inhibition of N₂ fixation caused by excessive O₂ within the symbiosomes.Abbreviations Lb leghaemoglobin - LbO₂ oxyleghaemoglobin - [O₂] concentration of free, dissolved O₂ - Y fractional oxygenation of Lb - Y _av volume averagedY     

The Site of Oxygen Limitation in Soybean Nodules

In legume nodules the [O₂] in the infected cells limits respiration and nitrogenase activity, becoming more severe if nodules are exposed to subambient O₂ levels. To identify the site of O₂ limitation, adenylate pools were measured in soybean (Glycine max) nodules that were frozen in liquid N₂ before being ground, lyophilized, sonicated, and separated on density gradients of nonaqueous solvents (heptane/tetrachloroethylene) to yield fractions enriched in bacteroid or plant components. In nodules maintained in air, the adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) was lower in the plant compartment (0.65 ± 0.04) than in the bacteroids (0.76 ± 0.095), but did not change when the nodulated root system was exposed to 10% O₂. In contrast, 10% O₂ decreased the bacteroid AEC to 0.56 ± 0.06, leading to the conclusion that they are the primary site of O₂ limitation in nodules. To account for the low but unchanged AEC in the plant compartment and for the evidence that mitochondria are localized in O₂-enriched microenvironments adjacent to intercellular spaces, we propose that steep adenylate gradients may exist between the site of ATP synthesis (and ADP use) in the mitochondria and the extra-mitochondrial sites of ATP use (and ADP production) throughout the large, infected cells.