Detopping causes production of intercellular space occlusions in both the cortex and infected region of soybean nodules

A structural analysis was conducted to determine whether glycoprotein‐containing intercellular space occlusions are involved in medium‐term regulation of O₂ diffusion in soybean (Glycine max) nodules. Alterations in O₂ diffusion were induced by a 3 h detopping treatment, and glycoprotein was immunolocalized with the monoclonal antibodies MAC236 and MAC265. Western blots of unstressed nodules revealed that these antibodies recognize antigens with two different molecular weights in soybean nodules. Tissue printing of halved nodules showed that both antigens were present in fresh nodules from control and 3 h detopped plants. The main localization appeared to be the inner cortex, but some immunolabelling also occurred in the infected region. ELISAs demonstrated a significant increase in total nodule concentration of intercellular glycoprotein following detopping, and cryosections of fresh nodules from this treatment also showed localization of antigens within the intercellular spaces of the infected region. The production of intercellular space occlusions in both the mid‐cortex and infected regions after 3 h detopping was confirmed by light microscopy and silver‐enhanced immunolabelling; cortical changes were quantified by image analysis techniques. Electron microscopy revealed that the occlusions within the infected region were less dense and less heavily labelled than those in the cortex. These results are discussed in relation to O₂ diffusion regulation in soybean nodules     

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.     

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.     

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.     

An osmotic hypothesis for the regulation of oxygen permeability in soybean nodules

Nodule permeability (P) controls the amount of O₂ entering the nodule, and thereby the rates of both nodule respiration and N₂ fixation. P may be regulated by changes in the effective thickness of a water-filled diffusion barrier in the nodule cortex. Regulation of diffusion barrier thickness was hypothesized to result from changes in the water content of intercellular spaces. Modulation of intercellular water would be a response to osmotic potential gradients in the tissue. To test this hypothesis, preliminary experiments examined three classes of solutes (soluble sugars, free amino acids, and ureides) in nodules of intact plants exposed to 10 or 21 kPa O₂ for 24 h. Neither soluble sugars nor free amino acids in nodules were responsive to O₂ treatments. However, nodule ureides accumulated after exposure to 10kPa O₂ for 24 h. A symplastic increase in nodule ureides under the 10kPa O₂ treatment compared to the 21 kPa O₂ treatment may have removed water from intercellular spaces in the nodule cortex and increased P. In addition, the nodule cortex of intact plants was infiltrated with water, polyethylene glycol (PEG), KC1, or Na-succinate solutions to determine the effect of intercellular water and osmoticants on dinitrogenase activity and P. Results from infiltrating the apoplast of the nodule cortex with osmotic solutions indicated that both increases in intercellular water and decreases in the apoplastic water potential decrease dinitrogenase activity and P. Furthermore, the inability to recover dinitrogenase activity and P following the infiltration of the cortex with PEG compared to either KCl or Na-succinate treatments may indicate that recovery was dependent upon removal of the solute from the apoplast.     

Effect of localized nitrogen availability to soybean half-root systems on photosynthate partitioning to roots and nodules

Soybean (Glycine max [L.] Merr. cv Davis) was grown in a split-root growth system designed to maintain control of the root atmosphere. Two experiments were conducted to examine how 80% Ar:20% O₂ (Ar:O₂) and air (Air) atmospheres affected N assimilation (NH₄NO₃ and N₂ fixation) and the partitioning of photosynthate to roots and nodules. Application of NH₄NO₃ to nonnodulated half-root systems enhanced root growth and root respiration at the site of application. A second experiment applied Ar:O₂ or air to the two sides of nodulated soybean half-root systems for 11 days in the following combinations: (a) Air to both sides (Air/Air); (b) Air to one side, Ar:O₂ to the other (Air/Ar:O₂), and (c) Ar:O₂ to both sides (Ar:O₂/Ar:O₂). Results indicated that dry matter and current photosynthate (¹⁴C) were selectively partitioned to nodules and roots where N₂ was available. Both root and nodule growth on the Air side of Air/Ar:O₂ plants was significantly greater than the Ar:O₂ side. The relative partitioning of carbon and current photosynthate between roots and nodules on a half-root system was also affected by N₂ availability. The Ar:O₂ sides partitioned relatively more current photosynthate to roots (57%) than nodules (43%), while N₂-fixing root systems partitioned 36 and 64% of the carbon to roots and nodules, respectively. The Ar:O₂ atmosphere decreased root and nodule respiration by 80% and nitrogenase activity by 85% compared to half-root systems in Air while specific nitrogenase activity of nodules in Ar:O₂ was 50% of nodules supplied Air. Results indicated that nitrogen assimilation, whether from N₂ fixation or inorganic sources, had a localized effect on root development. Nodule development accounted for the major decrease in total photosynthate partitioning to non-N₂-fixing nodules. Soybean compensates for ineffective nodulation by controlling the flux of carbon to ineffective nodules and their associated roots.     

Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

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.     

Observation of the Oxygen Diffusion Barrier in Soybean (Glycine max) Nodules with Magnetic Resonance Microscopy

The effects of selected gas perfusion treatments on the spinlattice relaxation times (T₁) of the soybean (Glycine max) nodule cortex and inner nodule tissue were studied with ¹H high resolution magnetic resonance microscopy. Three gas treatments were used: (a) perfusion with O₂ followed by N₂; (b) O₂ followed by O₂; and (c) air followed by N₂. Soybean plants with intact attached nodules were placed into the bore of a superconducting magnet and a selected root with nodules was perfused with the gas of interest. Magnetic resonance images were acquired with repetition times from 50 to 3200 ms. The method of partial saturation was used to calculate T₁ times on selected regions of the image. Calculated images based on T₁ showed longer T₁ values in the cortex than in the inner nodule during all of the gas perfusions. When nodules were perfused with O₂-O₂, there was no significant change in the T₁ of the nodule between the two gas treatments. When the nodule was perfused with O₂-N₂ or air-N₂, however, the T₁ of both the cortex and inner nodule increased. In these experiments, the increase in T₁ of the cortex was 2- to 3-fold greater than the increase observed in the inner nodule. A similar change in T₁ was found in detached live nodules, but there was no change in T₁ with selective gas perfusion of detached dead nodules. These observations suggest that cortical cells respond differently to selected gas perfusion than the inner nodule, with the boundary of T₁ change sharply delineated at the interface of the inner nodule and the inner cortex.     

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     

OXYGEN CONCENTRATION WITHIN THE NITROGEN-FIXING ROOT NODULES OF MYRICA GALE L.

Microelectrodes were used to study the oxygen concentration within Myrica gale L. nodules. Low oxygen concentrations were found only in the region of the mature, nitrogen-fixing endophyte, and appeared to correspond to clusters of infected host cells. The oxygen concentration in the remainder of the nodule was much higher. Interconnected intercellular air spaces were demonstrated by infiltration with India ink. Infiltration of the spaces with water greatly reduced oxygen concentration throughout the nodule, indicating that they function in supplying oxygen to the infected cells and remainder of the nodule. These results differ from those found previously for soybean nodules and provide evidence that legume and actinorhizal nodules have different mechanisms for protecting nitrogenase from oxygen.     

Temporal Relationships between Nitrogenase and Intercellular Glycoprotein in Developing White Lupin Nodules

The development of the N₂-fixing symbiosis between white lupin (Lupinus albusL.) cv. Multolupa andBradyrhizobiumstrain ISLU16 was followed using the acetylene reduction assay (ARA), immunoblots of protein extracts, and microscopy/immunogold labelling at 0, 8, 12, 17 and 20 d after infection. There was no ARA at 0, 8 and 12 d, although macroscopically visible nodule primordia had formed on roots by 8 d. The lack of nitrogenase at these times was confirmed by a negative signal to immunogold labelling with nitrogenase-specific antibodies. At 17 d three out of six plants had ARA, and nodules from these gave a positive signal with the nitrogenase antibody. By contrast, ARA⁻(fix⁻) nodules at 17 d were smaller (mean radius of 0.49 mm compared to 1.01 mm with fix⁺nodules) and gave a negative signal with the nitrogenase antibody. Western blots of nodule protein extracts using the monoclonal antibodies MAC236 and MAC265 (which recognize two epitopes on a glycoprotein which is considered to be involved in both rhizobial infection and the regulation of nodule oxygen diffusion) gave a strong signal with nodules (fix⁺) from 20 d plants and with 17 d fix⁺plants. The signal with MAC236/MAC265 was substantially weaker with nodules from 17 d fix⁻plants, and there was no signal apparent from nodules/nodulated roots from the 0, 8 and 12 d harvests. However, further investigation using immunogold labelling revealed that not only were MAC236 and MAC265 expressed within cortical intercellular spaces in 20 d and 17 d fix⁺/fix⁻nodules, but they were also strongly expressed in the developing cortex surrounding the newly-infected tissue in 8 d nodules, as well as in intercellular spaces within the cortex and infected tissue of 12 d nodules. These data demonstrate that the glycoprotein recognized by MAC236 and MAC265 is present before the onset of nitrogenase expression and function, but expression of the epitopes appears to be enhanced from the onset of N₂fixation. Nodules at all harvests were investigated for the presence of infection threads, as the MAC236/MAC265-recognized glycoprotein is also a component of the infection thread matrix in nodules from other legumes. Infection threads were not seen in nodules from any of the harvests except for the 20 d nodules, and then only after serial sectioning. The latter revealed occasional short wide infection threads entering and releasing rhizobia into small pockets of uninfected cells, within the infected tissue, but not within the meristems. The matrix of these infection threads labelled weakly, or not at all, with MAC236 and MAC265, and it was concluded that the majority of the MAC236/MAC265 detected in lupin nodule extracts originated from glycoprotein within cortical intercellular spaces.     

Diffusion and reaction of oxygen in the central tissue of ureide-producing legume nodules

Previous simulation models for the diffusion and reaction of oxygen in legume nodules were based on infected cells and neglected adjacent uninfected cells. This study uses a three-dimensional model of the central zone of legume nodules made up of the two cell types represented by a geometrically defined, space-filling, binary combination of polyhedra, each with bevelled edges to allow for a network of intercellular gas spaces. The model predicted a distinctively compartmentalized distribution of [O₂] between uninfected and infected cells; with high O₂ concentrations for an uninfected cell being consistent with, and necessary for, efficient operation of uricase and ureide synthesis and low O₂ concentrations across most of the infected cell providing a suitable environment for N₂-fixation. Compartmentalization of O₂ also predicted significant O₂ fluxes between cell types, compromising maintenance of low [O₂] in infected cells, as well as high [O₂] in uninfected cells. The results predict that there might be significant resistance to O₂ diffusion across the cell : cell interface due to the plasmalemma and cell walls.     

Symbiotic functioning,structural adaptation,and subcellular organization of root nodules from <Emphasis Type="Italic">Psoralea pinnata</Emphasis> (L.) plants grown naturally under wetland and upland conditions in the Cape Fynbos of South Africa

In the Cape Fynbos of South Africa, Psoralea pinnata (L.) plants occur naturally in both wetland and well-drained soils and yet effectively fix N₂ under the two contrasting conditions. In this study, nodule structure and functioning in P. pinnata plants from the two habitats were evaluated using light and transmission electron microscopy (TEM), as well as the ¹⁵N natural abundance technique. The results showed that, structurally, fully developed P. pinnata nodules were spherical in shape with six components (namely, lenticels, periderm, outer cortex, middle cortex, inner cortex, and a central bacteria-infected medulla region). Morphometric analysis revealed 44 and 84 % increase in cell area and volume of wetland nodules compared to those from upland. The percentage area of nodules occupied by the middle cortex in wetland nodules was twice that of upland nodules. As a result, the size of the medulla region in wetland nodules was significantly reduced compared to upland nodules. Additionally, the average area of medulla occupied by intercellular air spaces in wetland nodules was about five times that of upland nodules (about 431 % increase in wetland over upland nodules). TEM data also showed more bacteroids in symbiosomes of upland nodules when compared to wetland nodules. However, isotopic analysis of above-ground plant parts revealed no differences in symbiotic parameters such as N concentration, ?¹⁵N and %Ndfa between wetland and upland P. pinnata plants. These results suggest that, under limiting O₂ conditions especially in wetlands, nodules make structural and functional adjustments to meet the O₂ demands of N₂-fixing bacteroids.     

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.     

Morphological and structural adaptation of nodules of cowpea to functioning under sub- and supra-ambient oxygen pressure

Nodules of cowpea (Vigna unguiculata (L.) Walp. cv. Vita 3:Bradyrhizobium CB 756) from 28-d-old plants cultured for 23 d with their root systems maintained in O₂ levels from 1 to 80% (v/v, in N₂) in the external gas phase showed a range of structural changes which have been interpreted in relation to an over- or under-supply of O₂. A response to the partial pressure of O₂ in the gas phase (pO₂) was noted with respect to nodule size, lenticel development, the relative distributions of cortical and infected central tissue, the differentiation of cortex, especially the inner cortex, the frequency and size of infected and uninfected interstitial cells, the volume of extracellular spaces both in cortex and infected tissue, and in the frequency of bacteroids. As a consequence of these changes the surface area of inner cortex relative to the nitrogenase-containing units of fixing tissue (infected cells or bacteroids) was increased by as much as 20-fold. Effectiveness of bacteroid functioning increased from 0.10 ± 0.02 · 10^-9 μmol acetylene reduced per bacteroid in air-grown nodules to 0.9 ± 0.16 · 10^-9 (same units) per bacteroid in those cultured in 1% O₂. This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian International Development Assistance Bureau postgraduate fellowship (to F.D.D.). The authors wish to thank Dr. W.F.C. Blumer for his considerable help with morphometric analysis, Dr. J. Kuo for guidance in the use of histological techniques, and to Dr. J.S. Pate for the suggestion that lenticel development might be quantified by surface staining of nodules.     

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     

Adenylate-coupled ion movement. A mechanism for the control of nodule permeability to O2 diffusion

In response to changes in phloem supply, adenylate demand, and oxygen status, legume nodules are known to exercise rapid (seconds to hours) physiological control over their permeability to oxygen diffusion. Diffusion models have attributed this permeability control to the reversible flow of water into or out of intercellular spaces. To test hypotheses on the mechanism of diffusion barrier control, nodulated soybean (Glycine max L. Merr.) plants were exposed to a range of treatments known to alter nodule O2 permeability (i.e. 10% O2, 30% O2, Ar:O2 exposure, and stem girdling) before the nodules were rapidly frozen, freeze dried, and dissected into cortex and central zone (CZ) fractions that were assayed for K, Mg, and Ca ion concentrations. Treatments known to decrease nodule permeability (30% O2, Ar:O2 exposure, and stem girdling) were consistently associated with an increase in the ratio of [K+] in cortex to [K+] in the CZ tissue, whereas the 10% O2 treatment, known to increase nodule permeability, was associated with a decrease in the [K+]cortex:[K+](CZ). When these findings were considered in the light of previous results, a proposed mechanism was developed for the adenylate-coupled movement of ions and water into and out of infected cells as a possible mechanism for diffusion barrier control 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.     

Nitrogen nutrition and the development and senescence of nodules on cowpea seedlings

Cowpea (Vigna unguiculata (L.) Walp cv. Vita 3) seedlings inoculated with Rhizobium strain CB756 were cultured with their root systems maintained in air or in Ar: O₂ (80:20, v/v) during early nodule development (up to 24 d after sowing). Compared with those in air, seedlings in Ar:O₂ showed progressive N deficiency with inhibited shoot growth, reduced ribulose-1,5-bisphosphate carboxylase and total protein levels and loss of chlorophyll in the leaves. Nodule initiation, differentiation of infected and uninfected nodule tissues and the ultrastructure of bacteriod-containing cells were similar in the air and Ar: O₂ treatments up to 16 d after sowing. Thereafter the Ar: O₂ treatment caused cessation of growth and development of nodules, reduced protein levels in bacteroids and nodule plant cells, and progressive degeneration of nodule ultrastructure leading to premature senescence of these organs. Provision of NO ₃ ^- (0.1–0.2 mM) to Ar: O₂-grown seedlings overcame the abovementioned consequences of N₂ deficiency on nodule and plant growth, but merely delayed the degenerative effects of Ar: O₂ treatment on nodule structure and senescence. Treatment of Ar: O₂-grown seedlings with NO ₃ ^- greatly increased the protein level of nodules but the increase was largely restricted to the plant cell fraction as opposed to the bacteroids. By contrast, NO ₃ ^- treatment of air-grown seedlings increased protein of bacteroid and host nodule fractions to the same relative extents when compared with air-grown plants not supplemented with NO ₃ ^- . These findings, taken together with studies of the distribution of N in nodules of symbiotically effective plants grown from ¹⁵N-labeled seed, indicate that direct incorporation of fixation products by bacteroids may be a critical feature in the establishment and continued growth of an effective symbiosis in the cowpea seedling.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase