Root Nodule Enzymes of Ammonia Assimilation in Alfalfa (Medicago sativa L.) : DEVELOPMENTAL PATTERNS AND RESPONSE TO APPLIED NITROGEN

Nitrogenase-dependent acetylene reduction activity of glasshouse-grown alfalfa (Medicago sativa L.) decreased rapidly in response both to harvesting (80% shoot removal) and applied NO₃⁻ at 40 and 80 kilograms N per hectare. Acetylene reduction activity of harvested plants grown on 0 kilogram N per hectare began to recover by day 15 as shoot regrowth became significant. In contrast, acetylene reduction activity of all plants treated with 80 kilograms NO₃⁻-N per hectare and harvested plants treated with 40 kilograms NO₃⁻-N per hectare remained low for the duration of the experiment. Acetylene reduction of unharvested alfalfa treated with 40 kilograms N per hectare declined to an intermediate level and appeared to recover slightly by day 15. Changes in N₂-fixing capacity were accompanied by similar changes in levels of nodule soluble protein.     

Relationship between Ureide N and N(2) Fixation, Aboveground N Accumulation, Acetylene Reduction, and Nodule Mass in Greenhouse and Field Studies with Glycine max L. (Merr)

The relationship between ureide N and N₂ fixation was evaluated in greenhouse-grown soybean (Glycine max L. Merr.) and lima bean (Phaseolus lunatus L.) and in field studies with soybean. In the greenhouse, plant N accumulation from N₂ fixation in soybean and lima bean correlated with ureide N. In soybean, N₂ fixation, ureide N, acetylene reduction, and nodule mass were correlated when N₂ fixation was inhibited by applying KNO₃ solutions to the plants. The ureide-N concentrations of different plant tissues and of total plant ureide N varied according to the effectiveness of the strain of Bradyrhizobium japonicum used to inoculate plants. The ureide-N concentrations in the different plant tissues correlated with N₂ fixation. Ureide N determinations in field studies with soybean correlated with N₂ fixation, aboveground N accumulation, nodule weight, and acetylene reduction. N₂ fixation was estimated by ¹⁵N isotope dilution with nine and ten soybean genotypes in 1979 and 1980, respectively, at the V9, R2, and R5 growth stages. In 1981, we investigated the relationship between ureide N, aboveground N accumulation, acetylene reduction, and nodule mass using four soybean genotypes harvested at the V4, V6, R2, R4, R5, and R6 growth stages. Ureide N concentrations of young stem tissues or plants or aboveground ureide N content of the four soybean genotypes varied throughout growth correlating with acetylene reduction, nodule mass, and aboveground N accumulation. The ureide-N concentrations of young stem tissues or plants or aboveground ureide-N content in three soybean genotypes varied across inoculation treatments of 14 and 13 strains of Bradyrhizobium japonicum in 1981 and 1982, respectively, and correlated with nodule mass and acetylene reduction. In the greenhouse, results correlating nodule mass with N₂ fixation and ureide N across strains were variable. Acetylene reduction in soybean across host-strain combinations did not correlate with N₂ fixation and ureide N. N₂ fixation, ureide N, acetylene reduction, and nodule mass correlated across inoculation treatments with strains of Bradyrhizobium spp. varying in effectiveness on lima beans. Our data indicate that ureide-N determinations may be used as an additional method to acetylene reduction in studies of the physiology of N₂ fixation in soybean. Ureide-N measurements also may be useful to rank strains of B. japonicum for effectiveness of N₂ fixation.     

Response to drought stress of nitrogen fixation (acetylene reduction) rates by field-grown soybeans

The effects of drought stress on soybean nodule conductance and the maximum rate of acetylene reduction were studied with in situ experiments performed during two seasons and under differing field conditions. In both years drought resulted in decreased nodule conductances which could be detected as early as three days after water was withheld. The maximum rate of acetylene reduction was also decreased by drought and was highly correlated with nodule conductance (r = 0.95). Since nodule conductance is equal to the nodule surface area times the permeability, the relationship of these variables to both whole-plant and unit-nodule nitrogenase activity was explored. Drought stress resulted in a decrease in nodule gas permeability followed by decreases in nodule surface area when drought was prolonged. Under all conditions studied acetylene reduction on a unit-nodule surface area basis was highly correlated with nodule gas permeability (r = 0.92). A short-term oxygen enrichment study demonstrated nodule gas permeability may limit oxygen flux into both drought-stressed and well-watered nodules of these field-grown soybeans.     

Effect of Nitrate on Bradyrhizobium — Mungbean (Vigna radiata) Symbiosis in presence of Syringaldehyde

Bradyrhizoblum — mungbean (Vigna radiata) symbiosis was influenced by the application of nitrate in combination with.yrlngaldehyde. Application of nitrate alone at lower concentrations (2, 5 mM) caused a reduction at initial stage (10 DAS) while at later stage (35 DAS–45 DAS) enhanced the nodulation status (nodule number, nodule weight) and nodule efficiency (acetylene reduction activity). Higher concentration of nitrate (10 mM) caused a reduction in nodule number, nodule weight and acetylene reduction activity. Syringaldehyde alone improved the nodulation status and nodule efficiency while it acted synergistically when applied in combination with nitrate. These effects were also reflected in the biomass of plants.     

Effect of light, dark, and temperature on root nodule activity (acetylene reduction) of soybeans

The objectives of this study were (a): to define the effects of light, dark, and temperature on nodule activity (acetylene reduction), and (b) to establish the contributions of reserve carbohydrate and recent photosynthate to the support of nodule function. An in situ assay of nodule activity was developed for use with intact, hydroponically grown soybeans (Glycine max [L.] Merr. cv. Calland).     

Proteolytic Activity in Soybean Root Nodules : Activity in Host Cell Cytosol and Bacteroids throughout Physiological Development and Senescence

Root nodules were harvested from chamber-grown soybean (Glycine max L. Merrill cv Woodworth) plants throughout development. Apparent nitrogenase activity (acetylene reduction) peaked before seeds began to develop, but a significant amount of activity remained as the seeds matured. Nodule senescence was defined as the period in which residual nitrogenase activity was lost. During this time, soluble protein and leghemoglobin levels in the host cell cytosol decreased, and proteolytic activity against azocasein increased. Degradative changes were not detected in bacteroids during nodule senescence. Total soluble bacteroid protein per gram of nodule remained constant, and an increase in proteolytic activity in bacteroid extracts was not observed. These results are consistent with the view that soybean nodule bacteroids are capable of redifferentiation into free-living bacteria upon deterioration of the legume-rhizobia symbiosis.     

The effects of soybean cultivar,rhizobium strain and nitrate on plant growth,nodule mass and acetylene reduction rate

Summary The effects of twelve strains ofBradyrhizobium japonicum and ten cultivars of soybean (Glycine max (L.) Merr.) on plant and nodule weights, and acetylene reduction rates (33 to 41 days) were measured in the presence and absence of 6mM nitrate. No interactions between strains and cultivars were observed. Strain by nitrate interactions were found for plant and nodule weights, and acetylene reduction rates per gram of nodule. Cultivar by nitrate interactions were found for nodule weights, acetylene reduction rates per plant and per gram of nodule. Blackhawk with all strains, and all cultivars with strains 110 and CB 1809, seemed to be able to grow as well in the absence of nitrate (utilizing nodule fixation) as in its presence. The problems of identifying strains and cultivars with especially good nitrogen fixing ability in the presence of nitrate are discussed.     

Development of Bacteroids in Alfalfa (Medicago sativa) Nodules

The morphology, acetylene reduction capability, and nucleic acid content of bacteroids in different regions of alfalfa (Medicago sativa var. Buffalo) nodules were studied by electron microscopy, gas chromatography, and laser flow microfluorometry, respectively. Bacteroids in the nodule tips were small (1 to 2.5 micrometers in length), had low nucleic acid content, and contained distinct central nucleoids. These bacteroids were comparatively inactive in acetylene reduction in situ. Bacteroids in the middle regions of alfalfa nodules were greatly enlarged (5 to 7 micrometers in length), had relatively high nucleic acid content, and did not possess central nucleoids. The bacteroids were very active in acetylene reduction. Bacteroids in the basal nodule region also were enlarged and without distinct nucleoid regions, but had relatively low nucleic acid content and low in situ acetylene-reducing activity.     

Regulation of soybean nitrogen fixation in response to rhizosphere oxygen: I. Role of nodule respiration

Nitrogen fixation (acetylene reduction) rates of nodules on intact field-grown soybean (Glycine max) subjected to altered oxygen concentration (0.06-0.4 cubic millimeter per cubic millimeter) returned to initial rates during an 8-hour transitory period. Hydroponically grown soybean plants also displayed a transitory (1-4 hours) response to changes in the rhizosphere oxygen concentration after which the fixation rates returned to those observed under ambient oxygen concentrations. It was hypothesized that soybean nodules contain a regulatory mechanism which maintains a stable oxygen concentration inside nodules at a sufficiently low concentration to allow nitrogenase to function. A possible physiological mechanism which could account for this regulation is adjustment in nodule respiration activity such that nodule oxygen concentration and nitrogen fixation are maintained at stable levels. Experiments designed to characterize the non-steady-state oxygen response and to test for the presence of nodule respiratory control are presented. Non-steady-state acetylene reduction and nodule respiration (oxygen uptake) rates measured after alterations in the external oxygen concentration indicated that the regulatory mechanism required 1 to 4 hours to completely adjust to changes in the external oxygen concentration. Steady-state nodule respiration, however, did not respond to alterations in the rhizosphere oxygen concentration. It was concluded that soybean nodules can adjust to a wide range of rhizosphere oxygen concentrations, but the mechanism which controls nitrogen fixation rates does not involve changes in the nodule respiration rate.     

Utilization of aldehydes and alcohols by soybean bacteroids

Aldehydes, alcohols and acids were tested for their ability to support acetylene reduction and oxygen consumption by Rhizobium japonicum bacteroids isolated from soybean nodules. Several alcohols and aldehydes increased acetylene reduction and oxygen uptake. This is consistent with the concept that the plant nodule cytosol can metabolize carbohydrate via anaerobic fermentative pathways.     

Limitation of acetylene reduction (nitrogen fixation) by photosynthesis in soybean having low water potentials

The role of photosynthesis and transpiration in the desiccation-induced inhibition of acetylene reduction (nitrogen fixation) was investigated in soybean (Glycine max [L.] Merr. var. Beeson) using an apparatus that permitted simultaneous measurements of acetylene reduction, net photosynthesis, and transpiration. The inhibition of acetylene reduction caused by low water potentials and their aftereffects could be reproduced by depriving shoots of atmospheric CO₂ even though the soil remained at water potentials that should have favored rapid acetylene reduction. The inhibition of acetylene reduction at low water potentials could be partially reversed by exposing the shoots to high CO₂ concentrations. When transpiration was varied independently of photosynthesis and dark respiration in plants having high water potentials, no effects on acetylene reduction could be observed. There was no correlation between transpiration and acetylene reduction in the CO₂ experiments. Therefore, the correlation that was observed between transpiration and acetylene reduction during desiccation was fortuitous. We conclude that the inhibition of shoot photosynthesis accounted for the inhibition of nodule acetylene reduction at low water potentials.     

Comparison of twoPisum sativum nodulation mutants with their parental cultivar

In comparison with the parental cv. Finale the ‘RisfixC’ supernodulator exhibited higher, continuously increasing nodule number and fresh mass accumulation, but substantially lower individual nodule fresh mass, leghemoglobin concentration, and specific acetylene reduction activity of nodule tissue. There were no substantial differences between Finale and ‘RisfixC’ in total acetylene reduction, nodule leghemoglobin accumulation per nodulated root, total and specific CO₂ evolution from nodulated roots and gross CO₂ respiratory costs of acetylene reduction. The ‘RisfixC’ also exhibited a substantially lower plant dry mass production (by 30%), but nitrogen concentration in shoots and carotenoid concentration in leaf tissue were significantly higher by 33 and 14%, and the chlorophylla+b content insignficantly higher than in the parental cultivar. In contrast, the nodulation mutant ‘Risnod29’, exhibited a somewhat higher nodule fresh mass accumulation (by 21%) and individual nodule fresh mass (by 23%), total and specific acetylene reduction (by 49 and 19%) and a somewhat more rapid plant dry mass accumulation compared with the cv. Finale.     

Leaf Nitrate Reductase, d-Ribulose-1,5-bisphosphate Carboxylase, and Root Nodule Development of Genetic Male-Sterile and Fertile Soybean Isolines

The objectives of this study were to determine the effect of pod and seed development on leaf chlorophyll concentration, and on activities of leaf ribulose bisphosphate carboxylase, leaf nitrate reductase, and root nodule acetylene reduction in field-grown soybean (Glycine max [L.] Merr.). Two genetic male-sterile lines and their fertile counterparts (Williams and Clark 63) were compared in both 1978 and 1979. Two additional lines (Wells × Beeson and Wells × Corsoy) were compared in 1979.

The expression of male-sterile character was nearly complete as very little outcrossing due to insect pollinators was observed. Male-sterile plants showed a delayed late season decline in leaf chlorophyll content and ribulose bisphosphate carboxylase activity when compared with fertile plants. A slight delay in the loss of in vivo leaf nitrate reductase activity was also observed for male-sterile plants. Root nodule fresh weight and acetylene reduction activity declined slightly more rapidly for fertile lines than for male-sterile lines in both years with differences significant on the last two to three sampling dates as leaf loss occurred in the control plants.

Seed development was found to increase slightly, the rate of decline of metabolic activity in fertile lines compared with that of male-sterile lines. However, pod development was not an a priori requirement for leaf and root nodule senescence. Male-sterile plants also lost photosynthetic and nitrogen metabolic competence, but at a slower rate. These results support the concept that pod and seed development does not signal monocarpic senescence per se but rather affects the rate at which senescence occurs after flowering.

     

Nitrogen metabolism of soybeans: I. Effect of tungstate on nitrate utilization, nodulation, and growth

The effects of N source (6 mm nitrogen as NO₃⁻ or urea) and tungstate (0, 100, 200, 300, and 400 μm Na₂ WO₄) on nitrate metabolism, nodulation, and growth of soybean (Glycine max [L.] Merr.) plants were evaluated. Nitrate reductase activity and, to a lesser extent, NO₃⁻ content of leaf tissue decreased with the addition of tungstate to the nutrient growth medium. Concomitantly, nodule mass and acetylene reduction activity of NO₃⁻-grown plants increased with addition of tungstate to the nutrient solution. In contrast, nodule mass and acetylene reduction activity of urea-grown plants decreased with increased nutrient tungstate levels. The acetylene reduction activity of nodulated roots of NO₃⁻-grown plants was less than 10% of the activity of nodulated roots of urea-grown plants when no tungstate was added. At 300 and 400 μm tungstate levels, acetylene reduction activity of nodulated roots of NO₃⁻-grown plants exceeded the activity of comparable urea-grown plants.     

Enzymes of ammonia assimilation and ureide biosynthesis in soybean nodules: effect of nitrate

The effect of nitrate on N₂ fixation and the assimilation of fixed N₂ in legume nodules was investigated by supplying nitrate to well established soybean (Glycine max L. Merr. cv Bragg)-Rhizobium japonicum (strain 3I1b110) symbioses. Three different techniques, acetylene reduction, ¹⁵N₂ fixation and relative abundance of ureides ([ureides/(ureides + nitrate + α-amino nitrogen)] × 100) in xylem exudate, gave similar results for the effect of nitrate on N₂ fixation by nodulated roots. After 2 days of treatment with 10 millimolar nitrate, acetylene reduction by nodulated roots was inhibited by 48% but there was no effect on either acetylene reduction by isolated bacteroids or in vitro activity of nodule cytoplasmic glutamine synthetase, glutamine oxoglutarate aminotransferase, xanthine dehydrogenase, uricase, or allantoinase. After 7 days, acetylene reduction by isolated bacteroids was almost completely inhibited but, except for glutamine oxoglutarate aminotransferase, there was still no effect on the nodule cytoplasmic enzymes. It was concluded that, when nitrate is supplied to an established symbiosis, inhibition of nodulated root N₂ fixation precedes the loss of the potential of bacteroids to fix N₂. This in turn precedes the loss of the potential of nodules to assimilate fixed N₂.     

Nitrate inhibition of legume nodule growth and activity : I. Long term studies with a continuous supply of nitrate

The synthesis and accumulation of nitrite has been suggested as a causative factor in the inhibition of legume nodules supplied with nitrate. Plants were grown in sand culture with a moderate level of nitrate (2.1 to 6.4 millimolar) supplied continuously from seed germination to 30 to 50 days after planting. In a comparison of nitrate treatments, a highly significant negative correlation between nitrite concentration in soybean (Glycine max [L.] Merr.) nodules and nodule fresh weight per shoot dry weight was found even when bacteroids lacked nitrate reductase (NR). However, in a comparison of two Rhizobium japonicum strains, there was only 12% as much nitrite in nodules formed by NR⁻R. japonicum as in nodules formed by NR⁺R. japonicum, and growth and acetylene reduction activity of both types of nodules was about equally inhibited. In a comparison of eight other NR⁺ and NR⁻R. japonicum strains, and a comparison of G. max, Phaseolus vulgaris, and Pisum sativum, the concentration of nitrite in nodules was unrelated to nodule weight per plant or to specific acetylene reduction activity. The very small concentration of nitrite found in P. vulgaris nodules (0.05 micrograms NO₂⁻-N per gram fresh weight) was probably below that required for the inhibition of nitrogenase based on published in vitro experiments, and yet the specific acetylene reduction activity was inhibited 83% by nitrate. The overall results do not support the idea that nitrite plays a role in the inhibition of nodule growth and nitrogenase activity by nitrate.     

Time course of acetylene reduction in nodules of five actinorhizal genera

The rate of acetylene reduction was measured as a function of time after addition of 10% acetylene in Alnus, Casuarina, Ceanothus, Datisca, and Myrica. The maximum rate occurred after 45 to 60 seconds and was maintained for an additional 0.5 to 4 minutes before a decline in rate to 30 to 90% of the maximum. The rate then recovered to a value of 63 to 98% of the maximum. Removal of the shoot and lower roots did not affect nodule activity.     

Characterization of Azospirillum Isolated from Nitrogen-Fixing Roots of Harvested Sorghum Plants

Root segments of harvested sorghum plants had acetylene reduction activity ranging from 11 to 61 nmol of ethylene formed per h per g (dry weight). Five strains of Azospirillum brasilense sp. nov. were isolated from root segments.     

Effects of nitrogen dioxide and nitrate nutrition on nodulation, nitrogenase activity, growth, and nitrogen content of bean

The influence of nutrient nitrate level (0-20 millimolar) on the effects of NO₂ (0-0.5 parts per million) on nodulation and in vivo acetylene reduction activity of the roots and on growth and nitrate and Kjeldahl N concentration in shoots was studied in bean (Phaseolus vulgaris L. cv Kinghorn Wax) plants. Exposing 8-day old seedlings for 6 hours each day, for 15 days, to 0.02 to 0.5 parts per million NO₂ decreased total nodule weight at 0 and 1 millimolar nitrate, and nitrogenase (acetylene reduction) activity at all concentrations of nitrate. The pollutant had little effect on root fresh or dry weights. Shoot growth was inhibited by NO₂. The NO₂ exposure increased nitrate concentration in roots only at 20 millimolar nutrient nitrate. Exposure to NO₂ markedly increased Kjeldahl N concentration in roots but generally decreased that in shoots. The experiments demonstrated that nutrient N level and NO₂ concentration act jointly in affecting nodulation and N fixing capability, plant growth and composition, and root/shoot relationships of bean plants.     

Nodulation and Nitrogen Fixation in Cliffrose (Cowania mexicana var. stansburiana (Torr.) Jeps.)

Nodulation and acetylene reduction were demonstrated for cliffrose (Cowania mexicana var. stansburiana [Torr.] Jeps.) in a greenhouse trial. Nitrogen fixation was also verified with ¹⁵N. Seedlings were grown in a soil known to nodulate both bitterbrush species (Purshia tridentata (Pursh) D.C. and P. glandulosa [Curran]) to which a suspension of crushed bitterbrush nodules had been added. Bitterbrush seedlings grew faster and reduced more acetylene than cliffrose seedlings planted in the same pots, but the specific activity of the nodules and the nodule mass to root mass ratios were approximately the same.