A comparison of inhibition of French-bean and soybean nitrogen fixation by nitrate, 1% oxygen or direct assimilate deprivation

Inhibition by NO⁻₃ of acetylene reduction in bean ( Phaseolus vulgaris L. cv. Contender) and soybean ( Glycine max L. cv. Amsoy 71) was measured in parallel with nodule carbohydrate and nitrate metabolism. In bean the onset of inhibition of C₂H₂ reduction (6 h) coincided with decreased import of assimilates and a lowering of carbohydrate pools (sucrose, glucose and starch). Nitrate reductase (EC 1.6.6.1) activity was induced in all plant organs after 3 h but no nitrite was detected in the nodules. In soybean, nodule carbohydrate concentrations and import of assimilates into the nodules increased markedly between 6 to 24 h after supply of nitrate when the nitrogenase (EC 1.7.99.2) was progressively inhibited. High nitrate reductase activity was observed in the nodules and nitrites accumulated because of insufficient nitrite reductase activity. The nitrate-induced inhibition of nitrogenase was compared with the inhibition observed with low oxygen around the roots (1% O²) or with direct assimilate deprivation (girdling or decapitation). Soybean and bean appeared equally sensitive to these treatments as regards to acetylene reduction. The results are discussed in relation to the current hypotheses explaining nitrate-induced inhibition of dinitrogen fixation: assimilate deprivation or nitrite poisoning. Present data are in favour of the first for bean and of the second for soybean.     

Nitrate and nitrite reduction by alfalfa root nodules: Accumulation of nitrite in Rhizobium melioti bacteroids and senescence of nodules

Addition of NO⁻₃ rapidly induced senescence of root nodules in alfalfa ( Medicago sativa L. cv. Aragon). Loss of nodule dry matter began at the lowest NO⁻₃ concentration (10 m M ) but degradation of bacteroid proteins was only detected when nodules were supplied with NO⁻₃ concentrations above 20 m M .
Bacteroids from Rhizobium meliloti contained high specific activities of nitrate reductase (NR) and nitrite reductase (NiR). Both enzymes were presumably substrate-induced although substantial enzyme activities were present in the absence of NO⁻₃ Typical specific activities for soluble NR and NiR of bacteroids under NO⁻₃ free conditions were 1.2 and 1.4 μmol (mg protein)⁻¹h⁻¹, respectively. In the presence of NO⁻₃, the specific activity of NR was considerably greater than that of NiR, thus causing NO⁻₂ accumulation in bacteroids. Nitrite levels in the bacteroids were linearly correlated with specific activities of NR and NiR, indicating that NO⁻₂ is formed by bacteroid NR and that this NO⁻₂ in turn, induces bacteroid NiR. Accumulation of NO⁻₂ within bacteroids also indicates that NO⁻₂ inhibits nodule activity after feeding plants with NO⁻₃     

Biochemical characterization of nitrate and nitrite reduction in the wild-type and a nitrate reductase mutant of soybean

Enzyme activities involved in nitrate assimilation were analyzed from crude leaf extracts of wild-type (cv. Williams) and mutant ( nr₁ ) soybean [ Glycine max (L.) Merr.] plants lacking constitutive nitrate reductase (NR) activity. The nr₁ soybean mutant (formerly LNR-2), had decreased NADH-NR, FMNH₂-NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNH₂-NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH₂ as a reductant source. Nitrite reductase activity was normal in the nr₁ plant type in the presence of reduced methyl viologen. Assuming that constitutive NR is similar in structure to nitrate reductases from other plants, presence of xanthine dehydrogenase activity and loss of cytochrome c reductase activity indicated that the apoprotein and not the molybdenum cofactor had been affected in the constitutive enzyme of the mutant. Constitutive NR from urea-grown wild-type plants had 1) greater ability to use FMNH₂ as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NO₃⁻ and HCO₃⁻, compared with inducible NR from NO₃⁻-grown nr₁ plants. The presence in soybean leaves of a nitrate reductase with a pH optimum of 7.5 is contrary to previous reports and indicates that soybean is not an exception among higher plants for this activity.     

Nitrate and nitrite reduction in the plant fraction of alfalfa root nodules

The plant fraction of alfalfa ( Medicago sativa L. cv. Aragon) nodules contained both nitrate reductase (NR) and nitrite reductase (NiR). Specific activity of NADH-NR from the cytosol of nodules not treated with NO₃- was about 30 nmol (mg protein)^-1-h^-1 and was not basically affected by NO₃ addition. In contrast, typical specific activity for cytosolic NiR was 1.5 umol (mg protein)^-1h^-1 using methyl viologen as electron donor. This activity strongly increased with NO₃ concentration, probably due to substrate induction. Maximal activity was 3.5 μmol (mg protein)^-1h^-1 at 50 to 200 mM NO₃.
Estimates indicate that the contribution of cytosol to the overall NR and NiR activities of alfalfa nodules is distinctly different: less than 10% and about 70%, respectively. The increasing amounts of NO₂ accumulating in the cytosol upon NO₃, supply, and the different response to NO₃ of bacteroid and cytosolic NRs support the concept that most of this NO₂ comes from the bacteroids.     

Nitrate metabolism in roots and nodules of Vicia faba in response to exogenous nitrate

Activities of nitrate reduction enzymes, nitrate reductase activity (NRA) and nitrite reductase activity (NiRA) from roots and nodules of 5 mutant genotypes and one commercial cultivar (Alameda) of faba bean ( Vicia faba L. var. minor) grown in the presence of N₂ alone or with additional NO⁻₃ in the medium have been studied. A naturally occurring mutant (VFM109) with impaired ability to reduce nitrate in its nodules is described. All the other cultivars of V. faba showed nodule NRA, although the range was very wide, from almost negligible (VFM72) up to 2 μmol h⁻¹ (g FW)⁻¹. This activity was entirely of plant origin. Root NRA also ranged widely accross cultivars. However, the level of activity expressed as well as the response of NRA to nitrate followed a pattern opposite to that observed in nodules. Roots and nodules of all cultivars showed very high rates of NiRA, respectively 50 and 150-fold higher than NRA, thus precluding accumulation of nitrite in these tissues. Root enzymes were significantly stimulated by nitrate while negative (NRA) or little effect (NiRA) was found for nodules. Nitrate and nitrite reduction are carried out by inducible enzymes in roots of V. faba and by constitutive enzymes in nodules, indicating that there may be different forms of these enzymes in each tissue. Differences in the plant genotype were a major cause of the variability in nitrate and nitrite reduction by nodulated root systems of V. faba .     

Regulation of nitrate reductase in detached oat leaves by light and oxygen

Regulation of nitrate reductase (NR, EC 1.6.6.1) by oxygen concentration and light was studied in segments of oat ( Avena sativa L. cv. Suregrain) leaves, using the in vivo nitrate reductase assay. The activity of NR decreased after excision in either light or darkness; the addition of cycloheximide prevented this decrease. Treatments that increased tissue permeability (anoxia, Triton X-100) also increased NR activity. There was in general less NR activity in the light than in the dark and also less under aerobic (21–100% O₂) than under anaerobic (0.3% O₂) conditions. Treatments with antioxidants improved the activity in the light, but only at high O₂ levels (21–100% O₂).
The results suggest that NR may be regulated by inhibitory proteins synthesized in either light or darkness, by permeability changes and by light-induced oxidations that occur when O₂ is present. Oxygen may control the activity by stimulating the synthesis of inhibitory proteins in the light and in the dark and by promoting oxidation of SH-groups in the light.     

Distribution of nitrate reductase activity in Vicia faba: Effect of nitrate and plant genotype

The short term effect of NO₃⁻ (12 mM) on nitrate reductase (NR. EC 1.6.6.1) activity has been studied in the roots, nodules and leaves of different genotypes of Vicia faba L. at the end of vegetative growth. Root and leaf NR activity responded positively to NO₃⁻ while nodule activity, where detected, proved to he strongly inhibited. The withdraw of this NO₃⁻ from the solution consistently reduced activity in the roots and leaves but surprising, promoted a significant increase in nodule activity, which matched or surpassed that of control plants On the other hand, nodules developed in the presence of 8 mM NO₃⁻ expressed an on average 141% higher level of NR activity than did controls. This effect was observed even in nodules with negligible control activity. In any case, a naturally occurring mutant (VF17) lacking root and nodule NR activity is described. The results indicate that in V. faba. the effects of NO₃⁻ and plant genotype on NR activity depended on plant organ and time of NO₃⁻ application, hut the distribution of NO₃⁻ reduction through the plain was mainly dependent on plant genotype, and to a lesser extent on NO: supply and plant age.     

Effect of low nitrate supply to nodulated lucerne on time course of activites of enzymes involved in inorganic nitrogen metabolism

Plants of lucerne ( Medicago sativa L. cv. Aragón) inoculated with several strains of Rhizobium meliloti were supplied with a low level of nitrate (5 m M ). After 1 week, normalised nodule mass, obtained by dividing nodule weight by shoot weight, was decreased by one-fourth. This result closely paralleled the bacteroid protein content of nodules, whereas the cytosolic content remained constant. Nitrate reductase activity (NRA, EC 1.7.99.4) of bacteroids increased rapidly after nitrate supply, with actual rates being highly dependent on the Rhizobium strain. The expression of cytosolic NR (EC 1.6.6.1) also varied depending on the bacterial strain but was largely insensitive to nitrate feeding. Nitrite reductase activity (NiRA, EC 1.7.2.2) of either bacteroid or plant origin was independent of the R. meliloti strain. Activation occurred after 3 and 7 days, respectively, of nitrate feeding. Significant amounts of nitrite were obtained throughout the experimental period from buffered extracts of both bacteroids and cytosol of nodules. However, when these nodules were ground in the presence of inhibitors of enzyme activity, nitrite was only found in nodules containing strain 102-F-51 after 1 week of treatment. These results agree with the recent hypothesis that nitrite plays a role in a secondary stage of nodule damage by nitrate. We propose that NiRA rather than NRA can be used as an internal probe of nitrate access to the infected region of nodules.     

Main centers of nitrate and nitrite reduction in young and nodulated yellow lupine (<Emphasis Type="Italic">Lupinus luteus</Emphasis>)

Nitrate and nitrite reduction centers in non-nodulated and symbiotic yellow lupine were analyzed. In young seedlings, nitrate was exclusively accumulated in roots, which also was shown as the main nitrate reduction center. In contrast, leaves were shown to play a key role in nitrite reduction. A similar distribution of nitrate reductase (NR) and nitrite reductase was found in nodulated plants. However, in field conditions characterized by low nitrate content, a disproportionately high level of NR activity in nodules was also observed during all stages of symbiotic growth. This feature was confirmed in nitrate-fed hydroponic cultures. Nodule NR activity was one order of magnitude higher than in roots, in spite of the small stored nitrate pool found inside nodules. This suggests that nodule NR activity had been induced not by nitrate itself but indirectly. Since bacteroids were shown to be responsible for the vast majority of nodule NR activity, the plausible explanation of this effect seems to be a dissimilatory nature of rhizobial NR. Considering that environmental nitrate could cause hypoxia inside nodules, this is the proposed way of the observed nodule NR induction.     

Nitrate reductase and molybdenum cofactor in annual ryegrass as affected by salinity and nitrogen source

The influence of salinity on the activity of nitrate reductase (NR, EC 1.6.6.1) and the level of the molybdenum cofactor (MoCo) as affected by the source and concentration of nitrogen was studied in annual ryegrass ( Lolium multiflorum cv. Westerwoldicum). Plants grown in sand were irrigated with nutrient solution with an electrical conductivity of 2 or 11.2 dS m⁻¹, containing nitrogen (0.5 or 4.5 m M ) in the form of NH₄NO₃ or NaNO₃ Salinity-treated (11.2 dS m⁻¹) plants produced less biomass and more organic nitrogen while accumulating more NO⁻₃ than control plants. Increased nitrogen concentration in the irrigation solutions enhanced biomass and organic nitrogen production as well as NO⁻₃ accumulation irrespective of the electrical conductivity. Salinity inhibited shoot growth and increased shoot NR activity of plants receiving 4.5 m M NH₄NO₃ or NaNO₃. Similar effects were observed in roots of plants grown in 4.5 m M NaNO₃. Nitrate added to a complementation medium containing ryegrass MoCo and the NR apoprotein of Neurospora crassa mutant nit-1 stimulated the activity of the reconstituted NR (NADPH-nitrate reductase, EC 1.6.6.3). Increased salinity and nitrogen in the nutrient solutions caused an increase of MoCo content in roots and shoots. Similar results were observed for NR activity in the shoots. The increase of MoCo in response to salinity was more pronounced than that of NR, especially in the roots. We conclude that the pool size of MoCo in ryegrass is not constant, but varies in response to nutritional and environmental factors.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

In search of the mechanism of nitrate inhibition of nitrogenase activity in legume nodules: Recent developments

The mechanisms involved in the inhibition of nitrogenase activity in legume nodules by nitrate is unclear. This paper reviews and evaluates proposed mechanisms of this inhibition. Emphasis is placed on recent developments, which suggest that nitrate causes an O₂ limitation of nitrogenase activity. Several mechanisms that involve a nitrate-induced increase in resistance to O₃ diffusion in the nodule cortex are discussed.     

Author idex volume 36 (1986)

Abstract Nitrate reduction to ammonia by marine Vibrio species was studied in batch and continuous culture. In pH-controlled batch cultures (pH 7.4; 50 mM glucose, 20 mM KNO₃), the nitrate consumed accumulated to more than 90% as nitrite. Under these conditions, the nitrite reductase (NO⁻₂→ NH₃) was severely repressed. In pH-controlled continuous cultures of V. alginolyticus with glucose or glycerol as substrates ( D = 0.045 h⁻¹) and limiting N-source (nitrate or nitrite), nitrite reductase was significantly derepressed with cellular activities in the range of 0.7–1.2 μmol min⁻¹ (mg protein)⁻¹. The enzyme was purified close to electrophoretic homogeneity with catalytic activity concentrations of about 1800 nkat/mg protein. It catalyzed the reduction of nitrite to ammonia with dithionite-reduced viologen dyes or flavins as electron donors, had an M _r of about 50 000 (determined by gel filtration) and contained c-type heme groups (probably 4–6 per molecule).     

Effect of nitrate on nitrogen fixation and nodule carbohydrate and organic acid concentrations in pea mutants deficient in nitrate reductase

Nitrate inhibits symbiotic N₂ fixation and a number of hypotheses concerned with NO₃⁻ assimilation have been suggested to explain this inhibition. These hypotheses were tested using a pea ( Pisum sativum L. cv. Juneau) with normal nitrate reductase NR; (EC 1,6,6,4) activity and two mutants of cv. Juneau, A317 and A334, with impaired NR activity. The plants were inoculated with three strains of Rhizobium leguminosarum and grown for 3 weeks in N-free medium, followed by 1 week in medium supplemented with 0, 5 or 10 m M KNO₃ before harvesting. NO₃ was taken up at comparable rates by the parent and the mutants and accumulated in leaf and stem tissue of the latter. Acetylene reduction rates were inhibited similarly in both the parent and mutants in the presence of KNO₃ but there were differences among rhizobial strains. Starch concentration of the nodules decreased by 46% in the presence of KNO₃ and there were differences among rhizobial strains but not among pea genotypes. Malate and succinate accumulated in nodules in the presence of KNO₃. These data are not consistent with the photosynthate deprivation hypothesis as a primary mechanism for NO₃ inhibition of N₂ fixation since NO₃ affected the nodule carbohydrate composition of all three pea genotypes in a similar manner. The lack of correlation between NR activity and NO₃ inhibition of N₂ fixation suggests that NO₃ assimilation may be only indirectly involved in the inhibition phenomenon.     

Accumulation of γ‐aminobutyric acid in nodulated soybean in response to drought stress

Nitrogen fixation and nodule permeability to O₂ diffusion are decreased by drought stress. Since γ‐aminobutyric acid (GABA) synthesis is rapidly stimulated by a variety of stress conditions including hypoxia, it was hypothesized that decreased O₂ availability in nodules stimulates glutamate decarboxylase (GAD) activity (EC 4.1.1.15), thereby resulting in GABA accumulation. First, the amino acid composition of xylem sap was determined in plants subjected to soil water deficits. While the xylem sap concentration of several amino acids increased when the plant was subjected to a water deficit, the greatest increase was in GABA. GABA accumulation was examined in response to stress induced by hypoxia or the addition of polyethylene glycol (PEG) to the nutrient solution. The exposure of soybean nodules to hypoxia for 6 h enhanced the GABA concentration by 6‐fold, but there was no change in GABA concentration in response to the PEG treatment. No major changes in the in vitro GAD activity were measured in nodule cytosol or bacteroids. The present data do not support the hypothesis that decreased nodule O₂ permeability and a resulting O₂ deprivation inside nodules may stimulate in vitro GAD activity and thus GABA accumulation. However, the data could indicate a possible effect of hypoxia and drought stress on the in vivo activity of GAD.     

Effect of decreased irradiance on N and C metabolism in leaves and roots of maize

The effects of decreased irradiance on fresh and dry weight, root respiration, levels of carbohydrates and N-compounds, and extractable activities of enzymes involved in C and N metabolism were evaluated in maize ( Zea mays L. cv. Plauto) seedlings during the 7 days following transfer from 450 to 200 μmol m⁻² s⁻¹ PAR. The fresh weight of roots and stems, the initiation of new leaves, root respiration rate, and the accumulation of dry matter, soluble sugars, starch, malate and amino acids in both leaves and roots were strongly reduced at low irradiance. In contrast, the level of nitrate was increased in leaves and only marginally affected in roots. Leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity started to decrease after 24–34 h, whereas ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity and chlorophyll content were unaffected or only slightly reduced. In both leaves and roots, the adjustment of N metabolism to low irradiance occurred through a relatively rapid (30% after 10 h) and large (60% after 3 days) decrease of nitrate reductase (NR; EC 1.6.6.1) activity, followed by slower and smaller changes in the activity of nitrite reductase (EC 1.7.7.1), glutamine synthetase (EC 6.3.1.2) and NAD-dependent glutamate dehydrogenase (EC 1.4.1.2). We suggest that the preferential decrease of NR activity relative to other N-assimilating enzymes may be important for preventing the accumulation of toxic N-compounds like ammonia in both leaf and root tissues.     

Predicting leaf-level fluxes of O3 and NO2: the relative roles of diffusion and biochemical processes

Pollutants like O₃ and NO₂ enter leaves through the stomata and cause damage during reactions with components of biological cell membranes. The steady-state flux rates of these gases into the leaf are determined by a series of physical and biochemical resistances including stomatal aperture, reactions occurring within the cell wall and the ability of the leaf to remove the products of apoplastic reactions. In the present study, multiple regression models incorporating stomatal conductance, apoplastic and symplastic ascorbate concentrations, and nitrate reductase (NR) activities were generated to explain the observed variations in leaf-level flux rates of O₃ and NO₂. These measurements were made on the plant Catharanthus roseus (Madagascar periwinkle). The best-fit model explaining NO₂ flux included stomatal conductance, apoplastic ascorbate and NR activity. This model explained 89% of the variation in observed leaf fluxes and suggested physical resistances, reaction between NO₂ and apoplastic ascorbate, and the removal rate of nitrate (generated by reactions of NO₂ and water) from the apoplast all play controlling roles in NO₂ flux to leaves. O₃ flux was best explained by stomatal conductance and symplastic ascorbate explaining 66% of the total variation in leaf flux. Both models demonstrate the importance of measuring processes other than stomatal conductance to explain steady-state leaf-level fluxes of pollutant gases.     

NADH-dependent nitrate reductase from two-row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)⁻¹ min⁻¹ at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH₂-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent K_m for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent K_m for nitrate as well as V_max whereas it did not alter the K_m for NADH.     

Molecular approaches to the analysis of nitrate assimilation

Abstract. The application of molecular approaches such as mutant analysis and recombinant DNA technology, in conjunction with immunology, are set to revolutionize our understanding of the nitrate assimilation pathway. Mutant analysis has already led to the identification of genetic loci encoding a functional nitrate reduction step and is expected to lead ultimately to the identification of genes encoding nitrate uptake and nitrite reduction. Of particular significance would be identification of genes whose products contribute to regulatory networks controlling nitrogen metabolism. Recombinant DNA techniques are particularly powerful and have already allowed the molecular cloning of the genes encoding the apoprotein of nitrate reductase and nitrite reductase. These successes allow for the first lime the possibility to study directly the role of environmental factors such as type of nitrogen source (NO₃⁻ or NH₄⁺) available to the plant, light, temperature water potential and CO₂ and O₂ tensions on nitrate assimilation gene expression and its regulation at the molecular level. This is an important advance since our current understanding of the regulation of nitrate assimilation is based largely on changes of activity of the component steps. The availability of mutants, cloned genes, and gene transfer systems will permit attempts to manipulate the nitrate assimilation pathway.     

Carbon and nitrogen partitioning in young nodulated pea (wild type and nitrate reductase-deficient mutant) plants exposed to NH4NO3

Carbon and nitrogen partitioning was examined in a wild-type and a nitrate reductase-deficient mutant (A317) of Pisum sativum L. (ev. Juneau), effectively inoculated with two strains of Rhizobium leguminosarum (128C23 and 128C54) and grown hydroponically in medium without nitrogen for 21 days, followed by a further 7 days in medium without and with 5 mM NH₄NO₃. In wild-type symbioses the application of NH₄NO₃ significantly reduced nodule growth, nitrogenase (EC 1.7.99.2) activity, nodule carbohydrates (soluble sugars and starch) and allocation of [¹⁴C]-labelled (NO₃⁻, NH₄⁺, amino acids) in roots. In nodules, there was a decline in amino acids together with an increase in inorganic nitrogen concentration. In contrast, symbioses involving A317 exhibited no change in nitrogenase activity or nodule carbohydrates, and the concentrations of all nitrogenous solutes measured (including asparagine) in roots and nodules were enhanced. Photosynthate allocation to the nodule was reduced in the 128C23 symbiosis. Nitrite accumulation was not detected in any case. These data cannot be wholly explained by either the carbohydrate deprivation hypothesis or the nitrite hypothesis for the inhibition of symbiotic nitrogen fixation by combined nitrogen. Our result with A317 also provided evidence against the hypothesis that NO₃⁻ and NH₄⁺ or its assimilation products exert a direct effect on nitrogenase activity. It is concluded that more than one legume host and Rhizobium strain must be studied before generalizations about Rhizobium /legume interactions are made.