Nodule Nitrogenase Activity Research Articles

A stimulatory effect of low nitrate levels on nodulation, leghaemoglobin content, nitrogenase activity, and growth of mung-bean plants (Vigna radiata)

Mung beans (Vigna radiata) were grown in the field, and measurement of leghaemoglobin and nitrogenase activity of root nodules during plant growth showed a maximum 30 days after sowing, i.e., just when flowering began. Mung beans were also grown in hydroponic cultures with varying amounts of nitrate nitrogen and harvested at 30 day s. The extent of root nodulation, leghaemoglobin accumulation and nitrogenase activity in the nodules, and plant dry weights were found to be maximal at a low concentration (0.5 mM) of nitrate. Dry weights of shoots and roots (exclusive of nodules) and total chlorophyll content of leaves were also increased more by this low concentration than at higher concentrations of 1–3.5 mM. Nodule formation, including leghaemoglobin content and nitrogenase activity, declined with increased nitrate levels. At the highest nitrate levels studied (4.5 and 5 mM) no nodules were formed, but the shoot and root dry matter increased markedly. It is evident from the results that a minimal amount of nitrate nitrogen is essential for efficient symbiotic nitrogen fixation.

Organization and expression of leghaemoglobin genes.

Leghaemoglobin genes in soybean (Glycine max) are present as a moderately reiterated family of sequences. Since there are identical restriction site patterns of these sequences in DNA isolated from leaf, root, or nodule tissue, the data suggest that no major changes in the organization or methylation of leghaemoglobin genes occur during their induction. Cloned soybean leghaemoglobin-cDNA cross hybridized with RNA from root nodules of kidney bean (Phaseolus vulgaris), and to a lesser extent, of pea (Pisum sativum) indicating sequence homology in the leghaemoglobin genes of these species. Hybridization to the genomic DNA restriction fragments of two other species, Glycine soja and Vicia faba, also indicated interspecies sequence homologies. Several restriction fragments appear to be common to all species examined. The induction of these genes occurs following infection of the plant by Rhizobium and is independent of the appearance of nitrogenase activity in the nodules. The level of expression is, however, influenced by various mutations in Rhizobium that result in the development of ineffective (nonnitrogen fixing) nodules.

Entry of Oxygen and Nitrogen into Intact Soybean Nodules

The apparent nitrogenase activity of soybean root nodules is reduced approximately 70% upon nodule detachment. By increasing the partial pressure of oxygen in the assay vessel from approximately 0 2 to 0 5 atm, the nitrogenase activity of detached nodules is restored to the original attached level. On the other hand, maximal assayable nitrogenase activity of nodules attached to the intact soybean plant is achieved in the presence of 0-2-0-3 atm of oxygen. Exposure of attached nodules to 0-5 atm oxygen causes a decrease in nitrogenase activity seemingly due to inactivation of nitrogenase. These and other data suggest that, upon detachment, the entry of oxygen into the nodules is impaired. A model for the regulation of airflow into attached and detached nodules is presented. This model is also consistent with response of nodule activity to water stress.

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH4NO3 (50 to 250 μg N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N. Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.

Soil potassium effects on nitrogenase activity with associated nodule components of hairy vetch at anthesis

Abstract Acetylene reduction techniques are frequently utilized to estimate legume nodule nitrogenase activity levels. However, the known symbiotic nitrogen fixation reactions have no equivalent for the rapid permeastic transport of C2H4 reduced by nitrogenase of rhyzobial cells through the cortex tissues with the subsequent volatile excretion that is essential for GC quantitation procedures. The objective of this study was to determine interrelationships of nitrogenase (C2H2 reduction) with associated cytosol enzyme components from morphologically homologous nodules of Madison hairy vetch (Vicia villosa, Roth) at anthesis as influenced by soil potassium levels. The vetch plants were grown in a siliceous thermic Psammentic Paleustalf, Eufaula, and inoculated with Rhizobium leguminosarum Frank, ATCC 10314. Highly significant enhancement of nitrogenase activity progressed from quadratic to linear with increased soil K levels in time‐course samplings at 30, 60, 90 and 120 min. incubations at 27C. Means as C2...

Seasonal Patterns of Nitrate Reductase and Nitrogenase Activities in Phaseolus vulgaris L.

The patterns of nitrate reductase activity (NRA) in the leaves (in vivo assay) and root nodule nitrogenase activity (C(2)H(2) reduction) were investigated throughout the season in field-grown Phaseolus vulgaris plants.Maximal NRA (per g fresh weight) occurred at early stages of leaf development but total activity (per leaf) was maximal when the leaf reached full size. In mature plants, most NRA was associated with the upper leaves. Nitrogenase activity was initiated about 2 weeks after sowing, reached a maximum at flowering (5 weeks after sowing) and declined rapidly thereafter. Nitrogenase activity followed the pattern of nodule development. After flowering, P. vulgaris was apparently able to take up and assimilate NO(-) (3) as evidenced by the increase in NO(-) (3) content of the stem and the high levels of NRA in the leaves. Total plant NRA was maximal after flowering and addition of NH(4)NO(3) to the soil at flowering resulted in even higher levels of NRA through most of the pod-filling period, thus resulting in higher seed yields (59% over control).It is proposed that P. vulgaris can benefit from both N(2) fixation and NO(-) (3) assimilation and that nitrate reductase plays an important role in the assimilation of nitrogen after flowering.

Morphology and Nitrogenase Activity of Agar Cultures and Root Nodules Formed by D-Cycloserine-resistant Mutants of Rhizobium sp. Strain 32H1

Summary: One-step mutants of Rhizobium sp. strain 32h1 resistant to d-cycloserine showed substantial (≥ 90%) loss of asymbiotic (agar culture) nitrogenase activity but only partial (25 to 50%) loss of symbiotic (root nodule) nitrogenase activity. Two- and three-step resistant mutants showed a further decline or complete loss of both forms of nitrogenase activity. Since d-cycloserine inhibited the uptake of alanine by the parent strain and since the mutants possessed a defective transport system for alanine, it is suggested that the mutants are unable to concentrate d-cycloserine. Agar cultures of a one-step resistant mutant contained the three morphological types of Rhizobium present in the parent culture. In contrast, only one morphological type of Rhizobium was found in cultures of two- and three-step resistant mutants. Root nodules formed by a one-step resistant mutant on cowpea contained fewer bacteroid-filled plant cells than were present in nodules formed by the parent strain. Root nodules formed by two- and three-step resistant mutants contained only a small number of infected plant cells and in many of these rhizobia had not been released from infection threads. It is suggested that changes in the permeability of the Rhizobium cell wall, resulting in resistance to d-cycloserine, are responsible for the morphological changes and loss of nitrogenase activity in these mutants.

Effectiveness ofLotusRoot Nodules

When grown in a nutrient solution containing combined nitrogen (NH4NO3), Lotus pedunculatus and L. tenuis seedlings inoculated with a fast-growing strain of Rhizoblum (NZP2037) did neither develop root nodules nor develop flavolans in their roots. In contrast, the roots of nodulated seedlings growing in a nitrogen-free nutrient solution contained flavolans. Flavolan synthesis coincided with root nodule development on these plants. When added as a single dose, high concentrations of NH4NO3 (5 and 10 mg N per plant) stimulated the growth of L. pedunculatus plants but suppressed nodulation and nitrogen fixation. In contrast the continued supply of a low concentration of NH4NO3 (1·0 mg N d−1 per plant) stimulated nitrogen fixation by up to 500%. This large increase in nitrogen fixation was associated with a large increase in nodule fresh weight per plant, a doubling of nodule nitrogenase activity, and a lowering of the flavolan content of the plant roots. The close relationship between nitrogen deficiency, nodule development, and flavolan synthesis in L. pedunculatus meant that it was not possible (by nitrogen pretreatment of plants) to alter the ineffective nodule response of a Rhizobium strain (NZP2213) sensitive to the flavolan present in the roots of this plant.

Influence of ammonium chloride on the nitrogenase activity of nodulated pea plants (Pisum sativum).

A study was made on the short-term effect of ammonium ions on the nitrogenase activity of pea root nodules. Nodulated pea plants (Pisum sativum), having reached maximum acetylene-reducing activity, were supplied with NH4Cl (20 mM). Nitrogenase activity of intact plants, detached nodules, and isolated bacteroids was measured at differed time intervals. A significant drop (20 to 40%) in the acetylene-reducing activity of treated intact plants and their detached nodules was observed after 1 day. No drop in the nitrogenase activity of bacteroids (assayed aerobically, or anaerobically after treatment with ethylenediaminetetraacetic acid-toluene) occurred for 2 to 4 days after the addition of NH4+ to the plants, depending on cultural conditions. From these results it is concluded that the adverse effect of NH4+ on acetylene reduction by intact plants and detached nodules during the first 2 days is not due to a decrease in the amount of nitrogenase in the bacteroids. It is suggested that the effect has to be attributed to a reduced supply to the bacteroids of energy-delivery photosynthates.

EFFECTS OF HANDLING AND SURGICAL TREATMENTS ON NITROGENASE ACTIVITY IN ROOT NODULES OF ALNUS GLUTINOSA, WITH SPECIAL REFERENCE TO THE APPLICATION OF INDOLE‐ACETIC ACID

SUMMARYThe possible effects of applied indole‐acetic acid upon nitrogenase activity and respiration in alder nodules could not be distinguished from the effects of the manipulative procedures themselves. The extent to which plants were handled during the acetylene reduction assay had a large inhibitory effect upon the apparent nitrogenase activity of the nodules.

Effect of Temperature on the Nitrogenase Activity of Intact and Detached Nodules inLotusandStylosanthes

Temperature and plant age influenced the nitrogenase activity of Lotus and Stylosanthes nodules. Time course studies using nodulated plants in closed vials showed a decline in activity after 48 h; regassing with 10% (v/v) acetylene in air partially restored the activity. Transfer of plants from 15, 20, 25, and 30 °C to 40 °C immediately stopped activity; this was completely restored within 1 h after return to original temperatures. Detached nodules cultured on nitrogen-free agar medium exhibited a sucrose concentration (2–8%, w/v)-dependent nitrogenase activity at each temperature. With 6% sucrose prolonged activity, up to 96 h, was obtained. Decline in nitrogenase activity in detached nodules was due partly to exposure to air during excision and transfer to the medium.

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti.

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concetrations. Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.

Some effects of environmental stress on seed yield of cowpea (Vigna unguiculata (L.) walp.) cv. Prima

Separate experiments examined nodulation and seed yield of cowpea cv. Prima after (a) changes in the level of combined nitrogen from 25 to 0 or 60 ppm N, (b) cycles of wilting and rehydration, and (c) shading to ca 50% full daylight. Plants were grown in the simulated tropical environment of a plastics ‘bubble’ house and experienced these changes over the growth stages: emergence to first flower, first flower to mid pod-fill or mid pod-fill to maturity. Seed yields of nodulated plants were unaffected by combined nitrogen supply and almost double those of non-nodulated plants (100 g cf 56 g per plant)-due mainly to increases in pod number per plant and mean seed weight. Reducing the nitrogen level from 25 to 0 ppm N, especially between mid pod-fill and maturity, reduced seed yields of non-nodulated plants to 36 g per plant. At the first flowering stage, plants grown without combined nitrogen had nitrogenase activities less than 10% of those supplied with 25 ppm N; 60 ppm N at any stage of development more than halved nitrogenase activity when compared with plants supplied with 25 ppm. Repeated wilting prior to flowering markedly reduced seed yields compared with the unstressed controls (41 g cf 76 g per plant)-mainly by decreasing subsequent pod production. Nodule weight and nitrogenase activity per plant were also much reduced. Wilting after flowering did not reduce yield, and nitrogenase activity was less affected. Shading throughout, or from first flower onwards, reduced seed yield by about 25% because fewer pods were produced. All shading treatments significantly increased mean seed weight compared with unshaded controls (116–121 mg cf 105 mg).

Regulation of nitrogenase activity in soybean nodules by ATP and energy charge

Nonphosphorylating condition under anaerobiosis stopped nitrogenase activity in nodules of soybean ( Glycine max var. Chippewa) in less than three minutes and aeration quickly activated the enzyme. This stop-and-go treatment can be repeatedly applied on excised nodules, and a concomitant low-and-high ATP and energy charge (EC) was observed. After 2 minutes under anaerobiosis, nodule ATP and EC were decreased, respectively, to 20 and 40% of the control. These decreases were not as great with longer anaerobic treatments, and there was no change in the content of total adenosine phosphates. Oxygen enrichment (40%) stimulated the activity of nitrogenase by 2.5 fold in four minutes with a concomitant increase of ATP and EC by 40% and 14%, respectively, and an exhaustion of AMP. Longer treatments of oxygen enrichment lessened the initial effects. These findings indicate that ATP and energy charge probably regulate the activity of nitrogenase in vivo and an active adenylate kinase must be operating in the nodules to maintain an energy supply for the basal metabolism and for the nitrogenase under temporary stressed conditions.

The absence of oxidized leghemoglobin in soybean root nodules during nodule development

To prevent autoxidation leghemoglobin was extracted from soybean root nodules of different ages in an atmosphere of carbon monoxide. Spectrophotometric examination of the leghemoglobins revealed that, during the functional life of the nodules, leghemoglobin exists only in its reduced form. Therefore, the decline of nitrogenase activity in older root nodules cannot be attributed to the accumulation of oxidized leghemoglobin.

Effects of incubation temperature and oxygen tension on nitrogenase activity of legume root nodules

Acetylene reduction and H2 evolution by legume root nodules from several plant species depended on incubation temperature; some nodules were active from 2 to 40°C. Acetylene reduction rates differed between plant species, with maximum activity at temperatures between 20 and 30°C forVicia faba, V. sativa, Trifolium pratense, T. subterraneum, Medicago truncatula and soybean, at 35°C forM. sativa and at 40°C for cowpea. OnlyM. sativa and cowpea reduced substantial amounts at 37.5°C. Temperatures from 2 to 10°C only slightly lessened activity ofT. subterraneum andV. sativa nodules. Nitrogenase functioned at temperatures which prevent establishment of other aspects of the symbiosis. The rate of acetylene reduction was constant for several hours at temperatures below 15°C, and activity continued for several days at 2°C for some species, but declined with time at warmer temperatures. Some nitrogenase was denatured at warmer temperatures, but the O2 tension in the assay vial also affected activity. In closed assay vessels nodule respiration decreased the pO2 and reduced nitrogenase activity. Activity was restored by adding O2 or regassing assay vials with air or Ar/O2. When the pO2 was maintained, acetylene reduction and H2 evolution by detached soybean nodules continued unchanged for 6 h.

Elephant allergy.

<h3>Summary</h3> • Copper is an essential nutrient for symbiotic nitrogen fixation. This element is delivered by the host plant to the nodule, where membrane copper transporter would introduce it into the cell to synthesize cupro-proteins. • COPT family members in model legume <i>Medicago truncatula</i> were identified and their expression determined. Yeast complementation assays, confocal microscopy, and phenotypical characterization of a <i>Tnt1</i> insertional mutant line were carried out in the nodule-specific <i>M.truncatula</i> COPT family member. • <i>Medicago truncatula</i> genome encodes eight COPT transporters. <i>MtCOPT1</i> (<i>Medtr4g019870</i>) is the only nodule-specific <i>COPT</i> gene. It is located in the plasma membrane of the differentiation, interzone and early fixation zones. Loss of MtCOPT1 function results in a copper-mitigated reduction of biomass production when the plant obtains its nitrogen exclusively from symbiotic nitrogen fixation. Mutation of <i>MtCOPT1</i> results in diminished nitrogenase activity in nodules, likely an indirect effect from the loss of a copper-dependent function, such as cytochrome oxidase activity in <i>copt1-1</i> bacteroids. • These data are consistent with a model in which MtCOPT1 transports copper from the apoplast into nodule cells to provide copper for essential metabolic processes associated with symbiotic nitrogen fixation.

Reduction of acetylene by nodules of Trifolium subterraneum as affected by root temperature, Rhizobium strain and host cultivar

The nitrogenase activity (measured by reduction of C2H2 to C2H4) of nodules of Trifolium subterraneum grown at root temperatures from 7°C–19°C was broadly correlated with nitrogen fixation. Root temperature did not affect enzyme activity per se but did affect the amount of enzyme formed. Exposure of nodules to 7°C for 24 h did not decrease activity cf. 19°C. Activity was greatest when nodules were about 4 days old, before swollen bacteroid forms were produced, and then declined. The effectiveness of a bacterial strain at a given temperature was related to the amount of enzyme produced and to its persistence. Nitrogenase activity should be measured throughout the plant growth cycle for valid comparisons of strain effectiveness.