Nodule Physiology Research Articles

Nodules from Fynbos legume Virgilia divaricata have high functional plasticity under variable P supply levels

Legumes have the unique ability to fix atmospheric nitrogen (N2) via symbiotic bacteria in their nodules but depend heavily on phosphorus (P), which affects nodulation, and the carbon costs and energy costs of N2 fixation. Consequently, legumes growing in nutrient-poor ecosystems (e.g., sandstone-derived soils) have to enhance P recycling and/or acquisition in order to maintain N2 fixation. In this study, we investigated the flexibility of P recycling and distribution within the nodules and their effect on N nutrition in Virgilia divaricata Adamson, Fabaceae, an indigenous legume in the Cape Floristic Region of South Africa. Specifically, we assessed tissue elemental localization using micro-particle-induced X-ray emission (PIXE), measured N fixation using nutrient concentrations derived from inductively coupled mass-spectrometry (ICP-MS), calculated nutrient costs, and determined P recycling from enzyme activity assays. Morphological and physiological features characteristic of adaptation to P deprivation were observed for V. divaricata. Decreased plant growth and nodule production with parallel increased root:shoot ratios are some of the plastic features exhibited in response to P deficiency. Plants resupplied with P resembled those supplied with optimal P levels in terms of growth and nutrient acquisition. Under low P conditions, plants maintained an increase in N2-fixing efficiency despite lower levels of orthophosphate (Pi) in the nodules. This can be attributed to two factors: (i) an increase in Fe concentration under low P, and (ii) greater APase activity in both the roots and nodules under low P. These findings suggest that V. divaricata is well adapted to acquire N under P deficiency, owing to the plasticity of its nodule physiology

Standardized mapping of nodulation patterns in legume roots

Optimizing nodulation in legumes is a target for crop improvement, and the spatial control of nodulation is just beginning to be unravelled. However, there is currently no method for standard phenotyping of nodulation patterns. Here we present a method and software for the quantitative analysis of nodulation phenotypes. Roots of nodulated peas (Pisum sativum), wild-type and two mutants, were photographed. Data from the photographs were extracted using custom image and data analysis software. The software makes it possible to extract each nodule's position along primary and lateral roots, and to represent the nodulated root system in a standardized way independent of the way roots are arranged in the soil. A wide variety of nodulation and root variables are calculated, and average spatial nodulation patterns can be computed from multiple samples. Standardized spatial analysis of nodulation patterns opens the way for comparative analyses among genotypes of a single legume species, as here in pea. This approach could also be used to compare nodulation patterns among crops, among plants grown under different environmental conditions, or among plants exposed to different pharmacological treatments. The proposed method should therefore prove useful for studies on nodule organogenesis and nodule physiology and for optimizing nodulation in crops.

Soybean ureide transporters play a critical role in nodule development, function and nitrogen export

Legumes can access atmospheric nitrogen through a symbiotic relationship with nitrogen-fixing bacteroids that reside in root nodules. In soybean, the products of fixation are the ureides allantoin and allantoic acid, which are also the dominant long-distance transport forms of nitrogen from nodules to the shoot. Movement of nitrogen assimilates out of the nodules occurs via the nodule vasculature; however, the molecular mechanisms for ureide export and the importance of nitrogen transport processes for nodule physiology have not been resolved. Here, we demonstrate the function of two soybean proteins - GmUPS1-1 (XP_003516366) and GmUPS1-2 (XP_003518768) - in allantoin and allantoic acid transport out of the nodule. Localization studies revealed the presence of both transporters in the plasma membrane, and expression in nodule cortex cells and vascular endodermis. Functional analysis in soybean showed that repression of GmUPS1-1 and GmUPS1-2 in nodules leads to an accumulation of ureides and decreased nitrogen partitioning to roots and shoot. It was further demonstrated that nodule development, nitrogen fixation and nodule metabolism were negatively affected in RNAi UPS1 plants. Together, we conclude that export of ureides from nodules is mediated by UPS1 proteins, and that activity of the transporters is not only essential for shoot nitrogen supply but also for nodule development and function.

Expression and localization of a Rhizobium-derived cambialistic superoxide dismutase in pea (Pisum sativum) nodules subjected to oxidative stress.

Two phylogenetically unrelated superoxide dismutase (SOD) families, i.e., CuZnSOD (copper and zinc SOD) and FeMn-CamSOD (iron, manganese, or cambialistic SOD), eliminate superoxide radicals in different locations within the plant cell. CuZnSOD are located within the cytosol and plastids, while the second family of SOD, which are considered to be of bacterial origin, are usually located within organelles, such as mitochondria. We have used the reactive oxygen species-producer methylviologen (MV) to study SOD isozymes in the indeterminate nodules on pea (Pisum sativum). MV caused severe effects on nodule physiology and structure and also resulted in an increase in SOD activity. Purification and N-terminal analysis identified CamSOD from the Rhizobium leguminosarum endosymbiont as one of the most active SOD in response to the oxidative stress. Fractionation of cell extracts and immunogold labeling confirmed that the CamSOD was present in both the bacteroids and the cytosol (including the nuclei, plastids, and mitochondria) of the N-fixing cells, and also within the uninfected cortical and interstitial cells. These findings, together with previous reports of the occurrence of FeSOD in determinate nodules, indicate that FeMnCamSOD have specific functions in legumes, some of which may be related to signaling between plant and bacterial symbionts, but the occurrence of one or more particular isozymes depends upon the nodule type.

SOIL APPLIED COBALT AND COPPER ALTER THE RHIZOBIUM SYMBIOSIS AND GROWTH STATUS OF LABLAB PURPUREUS (L.) SWEET

A pot experiment was performed to investigate the effect of cobalt (Co) and copper (Cu) nutrition on nodulation, nodule activity and growth of Lablab purpureus plants growing in sandy clay loam soil collected from Toshka region in south of Egypt. The pots were given nutrient solution only once with addition of increasing levels of cobalt and copper (50, 100, 150 and 200 μg) as cobalt or copper sulfate solution. The present study has provided a new insight into the effect of cobalt and copper on nodulation and nodule physiology of Lablab purpureus (L.) Sweet (kashrangeeg) that has not yet been examined. Thus, the present work suggest that Co and Cu application is essential for the enhancement of nodulation, nodule activity and growth of Lablab purpureus plants growing in sandy clay loam soil deficient for theses microelements.

Progress on research on actinorhizal plants.

In recent years, our understanding of the plant side of actinorhizal symbioses has evolved rapidly. No homologues of the common nod genes from rhizobia were found in the three Frankia genomes published so far, which suggested that Nod factor-like molecules would not be used in the infection of actinorhizal plants by Frankia. However, work on chimeric transgenic plants indicated that Frankia Nod factor equivalents signal via the same transduction pathway as rhizobial Nod factors. The role of auxin in actinorhizal nodule formation differs from that in legume nodulation. Great progress has been made in the analysis of pathogenesis-related and stress-related gene expression in nodules. Research on nodule physiology has shown the structural and metabolic diversity of actinorhizal nodules from different phylogenetic branches. The onset of large-scale nodule transcriptome analysis in different actinorhizal systems will provide access to more information on the symbiosis and its evolution.

The argon-induced decline in nitrogenase activity commences before the beginning of a decline in nodule oxygen uptake

Replacement of N(2) by argon in the air around nodules directs nitrogenase electron flow in its total onto H(+) resulting in increased nodule H(2) evolution (total nitrogenase activity (TNA)). However, argon application induces a so-called argon-induced decline in nitrogenase activity (Ar-ID) connected with decreased nodule oxygen permeability. Consequently, TNA measurements tend to underestimate total nitrogenase activity. It is unclear whether the decline in oxygen diffusion into nodules induces the Ar-ID, or whether a decline in nitrogenase activity is followed by lower nodule O(2) uptake. The objective of the present work was to examine the time sequence of the decline in nodule H(2) evolution and O(2) uptake after argon application. In addition, the reliability of TNA values, taken as quickly as possible after the switch to Ar/O(2), was tested through comparative measurement of (15)N(2) uptake of the same plants. Short-term TNA measurements in an optimized gas exchange measurement system yielded reliable results, verified by parallel determination of (15)N(2) uptake. A five min application of Ar/O(2) was without effect on the subsequent H(2) evolution in ambient air. A parallel experiment on control plants revealed that a decrease in nodule oxygen uptake began several minutes after the onset of the decline in H(2) evolution. We conclude that the primary effect of the replacement of N(2) by argon differs from oxygen diffusion control. A gas exchange system allowing an immediate taking of TNA yields reliable results and does not disturb nodule activity. Gas exchange measurements provide a powerful tool for studying nodule physiology and should be combined with material from molecular studies.

Visualization of symbiotic tissue in intact root nodules of Vicia tetrasperma using GFP-marked Rhizobium leguminosarum bv. viciae

In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of Rhizobium leguminosarum bv. viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB . allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along z-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested laboratory model for pea cross-inoculation group has been demonstrated.

Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO2 production) and nitrogenase activity (H2 production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N2:O2, thus under N2-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N2 deprivation that occur in acetylene reduction assays and during extended Ar:O2 exposures in H2 assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O2 from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO2/H2. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO2 produced per 2e− transferred by nitrogenase to reduction of N2. The results ranged from 2.6 to 3.4 mol CO2 produced per 2e−. Carbon cost expressed as g C produced per g N reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N2-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.

Nodulation under salt stress of alfalfa lines obtained after in vitro selection for osmotic tolerance

The culture of pasture legumes is of special interest for the world's agriculture because it results in the production of high value protein accompanied by improvement of soil fertility, since they undergo symbioses with dinitrogen-fixing bacteria. Alfalfa is the most important forage crop for the arid and semi-arid areas, where increased salinity of irrigated fields is one of the major constraints that limit crop productivity. Recently, using in vitro selection, we obtained alfalfa genotypes with increased tolerance to PEG 6000, an agent that mimics drought. Here, we present studies on salt tolerance with adult plants of these alfalfa genotypes grown either on nitrate or diazotrophically at various non-lethal salt concentrations. Growth parameters and parameters related to nodule physiology and development have been analysed. All R lines had a higher salt tolerance than their explant source genotype (T1) in germination and growth on nitrate-containing medium. Under diazotrophic growth conditions, R1 and R3 were with higher relative salt tolerance than T1. Nodule biomass was depressed at high salt concentrations in all genotypes. This growth decline went in pair with changes in carbohydrate partitioning and in the hormonal balance.

Effect of Water Stress on Nodule Physiology and Biochemistry of a Drought Tolerant Cultivar of Common Bean (Phaseolus vulgarisL.)

Plants of EMGOPA-201, a drought tolerant cultivar of common bean(Phaseolus vulgaris), were maintained either at 90% soil field capacity (SFC) or stressed by reducing SFC to 70, 50 or 30% over a 10 d period. Plant dry weight was not affected by any of these treatments although the number and weight of nodules was reduced at 50 and 30% SFC. Nitrogenase activity, determined by the acetylene reduction assay (ARA), was also reduced, on a plant basis, at 50% SFC and was almost stopped at 30% SFC. The latter treatment caused a marked increase in nodule O2diffusion resistance and induced nodule senescence. A time-course analysis of the 10 d 30% SFC treatment showed a decrease in leaf water potential from -0.5 to -0.87 MPa by 8 d, with a cessation of dry weight increase after 3 d, when leaf water potential was -0.65 MPa. Proteins in the host plant fraction of nodules decreased to 50% of control values by 10 d and leghaemoglobin (Lb) content was also lower at this stage. The activity of sucrose synthase (SS) showed a 76% reduction between 3 and 6 d, whilst glutamate synthase (GOGAT) activity showed a 40% reduction. The activity of other key enzymes of carbon metabolism was also reduced after 10 d. Nodule sucrose content increased to double that of control nodules by 6 d, before declining back to control levels at 10 d. Starch content fell by 3 d and continued to fall throughout the stress period. The results are discussed in terms of drought tolerance strategies in relation to growth and metabolism in whole plants and nodules.

Effects of oxygen on nodule physiology and expression of nodulins in alfalfa

Early nodulin 2 (ENOD2) transcripts and protein are specifically found in the inner cortex of legume nodules, a location that coincides with the site of a barrier to O2 diffusion. The extracellular glycoprotein that binds the monoclonal antibody MAC236 has also been localized to this site. Thus, it has been proposed that these proteins function in the regulation of nodule permeability to O2 diffusion. It would then be expected that the levels of ENOD2 mRNA/protein and MAC236 antigen would differ in nodules with different permeabilities to O2. We examined the expression of ENOD2 and other nodule-expressed genes in Rhizobium meliloti-induced alfalfa nodules grown under 8, 20, or 50% O2. Although there was a change in the amount of MAC236 glycoprotein, the levels of ENOD2 mRNA and protein did not differ significantly among nodules grown at the different [O2], suggesting that neither ENOD2 transcription nor synthesis is involved in the long-term regulation of nodule permeability. Moreover, although nodules from all treatments reduced their permeability to O2 as the partial pressure of O2 (pO2) was increased to 100%, the levels of extractable ENOD2 and MAC236 proteins did not differ from those measured at the growth pO2, further suggesting that if these proteins are involved in a short-term regulation of the diffusion barrier, they must be involved in a way that does not require increased transcription or protein synthesis.

Methods for the continuous measurement of O2 consumption and H2 production by nodulated legume root systems

Two examples are provided of the use of these detectors together with the soyabean/USDA16 symThe details of two systems for measuring O 2 uptake biosis in a flow-through system furnished with and H 2 production in flow-through gas systems used an infrared gas analyser (IRGA) to measure CO 2 to study nodule physiology and biochemistry are pre- production. sented here. Both are constructed from commercially available fuel cells. Oxygen uptake measurements are Key words: Root nodules, nitrogen fixation, hydrogen based upon the differential signal from paired production, oxygen uptake, respiratory quotient. detectors exposed to sample and blank gas streams. Because of the very small signal5background ratio needed to detect O 2 uptake against atmospheric O 2 Introduction concentration, these detectors were mounted in ther- The measurement of gaseous inputs and outputs from mally controlled aluminium blocks designed to avoid nodulated legume roots represents a valuable non-invasive changes in back pressure. Also to avoid apparent con- tool for studying nodule physiology and biochemistry. To centration differences arising because of pressure dif- facilitate such studies open-ended flow-through systems ferences at the detectors it is important that sample, have been developed in which gas streams of defined control and calibration gas flow rates are the same. composition can be passed over the nodulated roots of Hydrogen detectors were mounted in an aluminium intact undisturbed plants sealed into appropriate conblock similar to that used for O 2 , although the require- tainers (Minchin et al., 1983; Layzell et al., 1989). The ments for temperature and back pressure control are eZuent gas from these containers can be analysed for much less stringent for this application. respiratory CO 2 production, H 2 evolution and O 2 uptake. The limit for differential detection of O 2 uptake in A brief overview of the significance of these measureair was about 25 ppm (1 mmol l’1) and for H 2 production ments, of previous analytical problems and of the design, 1 ppm (0.04 mmol l’1). Linearity checks for the two construction and function of two detector systems for detectors over the range 4‐90% O 2 and 15‐750 ppm measuring H 2 production and O 2 uptake is presented here.

Wavelength options for monitoring leghaemoglobin oxygenation gradients in intact legume root nodules

Nodule oximetry, based on spectrophotometric measurements of leghaemoglobin (Lb) oxygenation in intact nodules, has provided numerous insights into legume nodule physiology. Fractional oxygenation of Lb (FOL) has been monitored at various wavelengths, but comparisons among wavelengths have not been published previously. Changes in transmittance were monitored simultaneously at 660 nm and either 560 or 580 nm as FOL was manipulated by changing the O 2 concentration around nodules of Medicago sativa L. or Lotus corniculatus L. Video microscopy at 580 nm was used to generate two-dimensional maps of FOL gradients in intact nodules. In general, all three wavelengths gave similar results. Small discrepancies between 660 and 580 nm, sometimes seen in nodules with high O 2 permeability, may indicate interference by the ferric Lb peak at 625nm. A slightly longer wavelength, for example 670 nm, might be preferable. No significant discrepancies among wavelengths were seen in nodules whose O 2 permeability had been reduced by a 48 h exposure to 10 mM nitrate. Minor gradients in FOL were seen in nodules of M. sativa and Trifolium repens L. under air and steeper gradients could be induced by various treatments. The existence of these gradients indicates at least some restriction of long-range O 2 diffusion within the infected zone. The FOL maps do not have enough spatial resolution to measure gradients within infected cells.

Natural occurrence of mycotoxins in broad bean ( Vicia faba L.) Seeds and their effect on Rhizobium-legume symbiosis

Seeds of faba bean ( Vicia faba L.) cultivar Giza 3 were screened for the presence of mycotoxins. Eleven out of 100 samples were positive. Aflatoxins B 1 and B 2 were found in 7 samples with a mean concentration of 30 μg kg −1 seeds. Aflatoxins B 1 B 2, G 1 and G 2 and ochratoxin A were each detected twice in separate samples with a mean concentration of 25 and 20 μg kg −1, respectively. Mycotoxins at concentrations of 100 or 200 μg kg −1 soil significantly decreased nodule number, nodule fresh weight and total nitrogenase activity. This was translated into reductions in dry matter accumulation and nitrogen yield of the bean. Mycotoxins also suppressed specific nitrogenase activity, NADH-dependent glutamate dehydrogenase (NADH-GDH) as well as glutamate synthase (NADH-GOGAT) activities. In addition, mycotoxins inhibited synthesis of leghaemoglobin, carbohydrate and protein in the nodule cytosol. Of the mycotoxins tested, aflatoxin B 1 was the most toxic. The decline in nitorgenase activity and total N concentration in the plants could be attributed to mycotoxins interfering with normal nodule physiology and function.

Natural occurrence of mycotoxins in broad bean (Vicia faba l.) seeds and their effect on Rhizobium‐legume symbiosis

AbstractSeeds of faba bean cultivar Giza 3 were screened for natural presence of mycotoxins. Eleven out of 100 samples were contaminated. Aflatoxins B1 and B2 were found in 7 samples while aflatoxins B1, B2, G1 and G2 and ochratoxin A were each detected twice in separate samples. Mycotoxins at concentrations of 100 or 200 μg kg−1 soil significantly decreased nodule number, nodule fresh weight and total nitrogenase activity, leading to reductions in dry matter accumulation and nitrogen yield of the bean. Mycotoxins also suppressed specific nitrogenase activity. NADH‐dependent glutamate dehydrogenase (NADH‐GDH) as well as glutamate synthase (NADH‐GOGAT) activities. In addition, mycotoxins inhibited synthesis of leghaemoglobin, carbohydrated and protein in the nodule cytosol. Of the mycotoxins tested, aflatoxin B1 was the most toxic. The decline in nitrogenase activity and total N concentration in the plants could be attributed to mycotoxins interference with normal nodule physiology and function.

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-aerated water culture or vermiculite. Growth, nodulation, nitrogen fixation and nodule physiology were investigated

Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativa) plants

Susceptibility of alfalfa (Medicago sativa L. cv. Aragón) nodules and leaves to water stress has been investigated. Nodule acetylene reduction activity (ARA), leaf CO2 exchange rate (CER) as well as soluble protein, proline and total soluble sugar (TSS) contents were determined during drought. Water status was estimated as water potential (Ψw) and Relative water content (RWC) of the respective tissues. Maximum rates of ARA required higher Ψw than CER. Nodules had lower RWC for a given Ψw than leaves. Water stress reduced soluble protein content in both tissues; however, the decline in soluble protein content was detected at greater Ψw in nodules than in leaves. Proline and TSS increased in leaves and nodules, and again the threshold Ψw triggering such accumulation was higher in nodule tissues. Oior results suggest that alfalfa nodules are more susceptible to water shortage than leaves. Effects of accumulated TSS and proline upon leaf and nodule physiology are discussed in relation to protein stability (proline), pH control (proline) and osmotic adjustment (proiine and TSS). The TSS accumulation induced by water stress suggests that substrate shortage would not be the primary effect of drought on nodule activity.

Measurement of Legume Nodule Respiration and O2 Permeability by Noninvasive Spectrophotometry of Leghemoglobin

Physiological regulation of nodule gas permeability has a central role in the response of legumes to such diverse factors as drought, defoliation, and soil nitrate. A new method for quantifying nodule respiration and O(2) permeability, based on noninvasive spectrophotometry of leghemoglobin, was evaluated using intact, attached nodules of Lotus corniculatus. First, the relationship between nodule respiration (O(2) consumption) rate and internal O(2) concentration was determined from the rate of decrease in fractional oxygenation of leghemoglobin (FOL) under N(2). The rate of increase of FOL under 100% O(2) was then used to calculate nodule O(2) permeability, after correcting for respiration. Inactivation of nitrogenase by exposure to 100% O(2) for 15 minutes led to decreases in both permeability and O(2)-saturated respiration (V(max)), but the brief (<15 seconds) exposures to 100% O(2) required by the assay itself had little effect on either parameter. A gradual increase in external O(2) concentration from 20 to 40% resulted in a reversible decrease in permeability, but no change in V(max). The new method is likely to be useful for research on nodule physiology and might also be applicable to agronomic research and crop improvement programs.

Nodule Physiology of a Supernodulating Soybean (Glyine max) Mutant

The nodule physiology of a supernodulating, nitrate tolerant symbiosis soybean (Glycine max (L.) Merr.) mutant (nts382) was compared to that of its wild-type parent, cv. Bragg. Nodule number and mass were greater in nts382 than cv. Bragg and individual nodule mass, bacteroid and haem content, and acetylene reduction activity per g nodule were less. Acetylene reduction activity expressed per mg bacteroid protein was the same in the two genotypes. In median sections, the ratio of infected to total nodule area was smaller in nts382, infected cell size was smaller and there were fewer bacteroids per peribacteroid envelope. When inoculum dose was decreased from 109 to 103 viable cells per pot, nodule number on nts382 decreased approximately to that on cv. Bragg; nodule size, bacteroid and haem contents increased as did nodule acetylene reduction activity. Application of moderate levels of nitrate, which did not significantly affect symbiotic parameters of cv. Bragg or high inoculum nts382, stimulated nodule growth and nitrogenase activity of low inoculum nts382. A combination of nitrate and low inoculum levels enhanced nodule parameters of nts382 to the level usually seen with cv. Bragg. When supernodulated, plant dry weight of nts382 was less than that of Bragg; decreasing inoculum dose had no significant effect but nitrate application increased plant growth; nitrate plus low inoculum induced similar plant growth to that of cv. Bragg. Nodule carbohydrate content was similar in both genotypes but nodule and xylem sap ureide contents were higher in nts382. In general, nodules of supernodulated nts382 resembled under-developed cv. Bragg nodules; when supernodulation was avoided by using low inoculum doses, ,nts382 nodules resembled those of cv. Bragg. Nitrogen metabolism in the mutant seems to be disturbed, resulting in ureide accumulation.