Effect of temperature on h(2) evolution and acetylene reduction in pea nodules and in isolated bacteroids

Nitrogenase (EC 1.7.99.2) activity in pea (Pisum savitum) nodules formed after infection with Rhizobium leguminosarum (lacking uptake hydrogenase) was measured as acetylene reduction, H₂ evolution in air and H₂ evolution in Ar:O₂. With detached roots the relative efficiency, calculated from acetylene reduction, showed a decrease (from 55 to below 0%) with increasing temperature. With excised nodules and isolated bacteroids similar results were obtained. However, the relative efficiency calculated from H₂ evolution in Ar:O₂ was unaffected by temperature. Measurements on both excised nodules and isolated bacteroids showed a marked difference between acetylene reduction and H₂ evolution in Ar:O₂ with increased temperature, indicating that either acetylene reduction or H₂ evolution in Ar:O₂ are inadequate measures of nitrogenase activity at higher temperature.     

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH₄NO₃ on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteriods in the nodule tissue were studied. The addition of NH₄NO₃ decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [³⁵S]sulfate into the components I and II of nitrogenase was similarly not affected.The addition of NH₄NO₃ decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.     

Nitrogenase activity of pea bacteroids as affected by carbohydrates and ammonium chloride

Summary Regulation and efficiency of the nitrogen-fixing system of the rhizobium-pea symbiosis were investigated. Acetylene reduction of detached root nodules was measured with various substrates added. Succinate, fumarate and malate were most effective in stimulating nitrogenase activity; glucose, pyruvate and citrate were also active. Acetylene reducing activity of detached nodules was inhibited by the addition of NH₄Cl, irrespective of the substrate present. Nitrogenase activity of isolated bacteroids was not influenced by NH₄Cl.Respiration of detached nodules was not significantly stimulated by the addition of substrates. Ammonium chloride did not influence respiration. With detached nodules and isolated bacteroids a consumption of about 16 g of carbohydrate per g of nitrogen fixed could be calculated. Detached nodules produced more hydrogen relative to the acetylene reduced than did isolated bacteroids and intact plants.Results obtained indicate that the regulation of nitrogenase activity and the efficiency of substrate consumption depend on environmental conditions.     

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.     

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.     

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.     

Effect of proline on nitrogenase activity in symbiosomes from root nodules of soybean (Glycine max L.) subjected to drought stress

Experiments were carried out to investigate if drought stressaffects the ability of bacteroids from soybean (Glycine maxL.) root nodules to utilize proline and malate to support nitrogenaseactivity. The bacteroids were isolated in sub-ambient oxygenand nitrogenase activity was measured by acetylene reduction.Nitrogenase activity supported by proline was 8-fold higherin bacteroids from drought-stressed nodules than in bacteroidsfrom control nodules. In contrast to the results with prolinethere was no significant response to drought stress in the rateof bacteroid nitrogenase activity supported by malate. The effectof drought stress on transport of proline and malate acrossthe symbiosome membrane was investigated by incubation of symbiosomesisolated in sub-ambient oxygen with radioactive tracers. Droughtstress tended to increase the rate of proline uptake relativeto a minor decrease in malate uptake into symbiosomes in responseto drought. There was no indication of a saturable camer inthe symbiosome membrane for either substrate at concentrationsin the range 0.1-2 mM. The rate of malate uptake into symbiosomeswas twice as high as the rate of proline uptake at all substratelevels tested. The protein composition of the symbiosome membranewas altered in response to drought stress and these changesmay relate .to the permeability of the symbiosome membrane. Key words: Drought stress, nitrogenase activity, proline, soybean nodules, symbiosome membrane, transport     

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR⁻ 108, NR⁻ 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N₂-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR⁻ 108 and NR⁻ 303 contained neither nitrate reductase nor nitrite reductase activities.

Addition of 20 millimolar KNO₃ to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO₃ treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.

The addition of either KNO₃ or KNO₂ to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.

     

Nitrate Inhibition of Legume Nodule Growth and Activity : II. Short Term Studies with High Nitrate Supply

Soybean plants (Glycine max [L.] Merr) were grown in sand culture with 2 millimolar nitrate for 37 days and then supplied with 15 millimolar nitrate for 7 days. Control plants received 2 millimolar nitrate and 13 millimolar chloride and, after the 7-day treatment period, all plants were supplied with nil nitrate. The temporary treatment with high nitrate inhibited nitrogenase (acetylene reduction) activity by 80% whether or not Rhizobium japonicum bacteroids had nitrate reductase (NR) activity. The pattern of nitrite accumulation in nodules formed by NR⁺ rhizobia was inversely related to the decrease and recovery of nitrogenase activity. However, nitrite concentration in nodules formed by NR⁻ rhizobia appeared to be too low to explain the inhibition of nitrogenase. Carbohydrate composition was similar in control nodules and nodules receiving 15 millimolar nitrate suggesting that the inhibition of nitrogenase by nitrate was not related to the availability of carbohydrate.

Nodules on plants treated with 15 millimolar nitrate contained higher concentrations of amino N and, especially, ureide N than control nodules and, after withdrawal of nitrate, reduced N content of treated and control nodules returned to similar levels. The accumulation of N₂ fixation products in nodules in response to high nitrate treatment was observed with three R. japonicum strains, two NR⁺ and one NR⁻. The high nitrate treatment did not affect the allantoate/allantoin ratio or the proportion of amino N or ureide N in bacteroids (4%) and cytosol (96%).

     

The effect of waterlogging on the synthesis of the nitrogenase components in bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum

The effect of waterlogging of root nodules on nitrogenase activity and synthesis was studied in Pisum sativum inoculated with Rhizobium leguminosarum (strain PRE). It was shown that: 1. nitrogenase activity of intact pea plants was decreased by waterlogging, 2. this decrease was paralleled by a decline of the amount of active nitrogenase determined in toluene EDTA treated bacteroids, 3. SDS-polyacrylamide gel electrophoresis revealed that the amount of nitrogenase component II (CII) decreased by waterlogging while the amount of component I (CI) was not markedly affected, and 4. analysis of bacteroid proteins after ³⁵SO₄ labeling of pea plants showed that CII synthesis was repressed while CI synthesis continued indicating that the synthesis of CI and CII is regulated by independent mechanisms.     

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.     

Identification and Characterization of a Bacteroid-Specific Dehydrogenase Complex in Rhizobium leguminosarum PRE

In membranes of Rhizobium leguminosarum bacteroids isolated from nitrogen-fixing pea root nodules, two different protein complexes with NADH dehydrogenase activity were detected. One of these complexes, with a molecular mass of 110 kilodaltons, was also found in membranes of free-living rhizobia, but the other, with a molecular mass of 550 kilodaltons, appeared to be present only in bacteroids. The bacteroid-specific complex, referred to as DH1, probably consists of at least four different subunits. Using antibodies raised against the separate polypeptides, we found that a 35,000-molecular-weight polypeptide (35K polypeptide) in the DH1 complex is bacteroid specific, while the other proposed subunits were also detectable in cytoplasmic membranes of free-living bacteria. Dehydrogenase complex DH1 is also present in bacteroids of a R. leguminosarum nifA mutant, indicating that the synthesis of the dehydrogenase is not dependent on the gene product of this nif-regulatory gene. A possible involvement of the bacteroid-specific DH1 complex in electron transport to nitrogenase is discussed.     

The effects of gibberellins and mepiquat chloride on nitrogenase activity in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA₃) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA₃ or PIX. GA₃ treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA₃-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA₃ and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA₃ treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA₃, respectively, in B. japonicum.     

Respiration supported nitrogenase activity of isolated Rhizobium meliloti bacteroids

Bacteroids having a high level of respiration-supported nitrogenase activity were isolated from nitrogen-fixing alfalfa root nodules. Gentle maceration under anaerobic conditions in the presence of sodium succinate and a fatty acid scavenging agent were employed in this method. A large proportion of isolated bacteroids retained a triple membrane structure as shown by transmission electron microscopy. Dicarboxylic acids of the TCA cycle (malate, fumarate, succinate), but not glutamate or aspartate, supported sufficient respiratory activity to supply the nitrogenase system with ATP and reducing equivalents and to protect the nitrogenase system from inactivation by 4% oxygen over a period of 20-30 min. Sugars did not support nitrogenase activity in intact bacteroids. The properties of the isolated bacteroids were ascribed to minimal damage to the cytoplasmic membrane and peribacteroidal membrane during isolation. With succinate as substrate and oxygen as terminal electron acceptor, initial nitrogenase activity was determined at 4% oxygen in the gas phase of the assay system employed. At this oxygen concentration, the sustained rate of acetylene reduction by respiring bacteroids was linear up to 30 min. Bacteroid activity declined rapidly with time of exposure to oxygen above 4% in the gas phase. The optimum temperature range for this activity was 10-20 degrees C. Nitrogenase activity was measurable at incubation temperatures below 10 degrees C under 4% oxygen. Functionally intact bacteroids had little nitrogenase activity under anaerobic conditions in the presence of an external source of ATP and reductant. Treatment of the bacteroids with chlorpromazine eliminated respiration-supported activity and rendered the bacteroid cell membrane permeable to external ATP. Bacteroids treated with chlorpromazine had high acetylene reducing activity with external ATP and dithionite in the absence of oxygen.     

Bacteroids Isolated from Ineffective Nodules of Pisum sativum Mutant E135 (syml3) Lack Nitrogenase Activity but Contain the Two Protein Components of Nitrogenase

Pea mutant E135 (sym15) forms ineffective (Fix^–) nodulesthat lack nitrogen fixing activity. To determine the developmentalstep blocked in E135 nodules we studied the nitrogenase activitiesin isolated bacteroids and in cell-free extracts of bacteroids,and measured the two components of nitrogenase protein in bacteroids.Bacteroids prepared anaerobically from E135 nodules showed noacetylene reduction activity in the presence and absence ofmyoglobin. Furthermore, no acetylene reduction activity by cell-freeextracts of E135 bacteroids was detected in the presence ofATP-generating system and dithionite. However, immuno-blottinganalyses revealed the presence of nitrogenase components I andII in E135 nodule bacteroids. These results suggest that a hostplant gene is involved in the expression of nitrogenase activityin symbiotic bacteria. (Received May 11, 1998; Accepted August 7, 1998)     

Effect of nitrate on the organic Acid and amino Acid composition of legume nodules

Nitrate supplied to legume plants inhibits the activity of nitrogenase in Rhizobium bacteroids in root nodules. The accumulation of amino N which is known to occur in Glycine max (L.) Merr. nodules as nitrogenase activity declines was studied in more detail by analysis of changes in free amino acid composition in response to high nitrate supply. A 6-fold increase in asparagine concentration in Bradyrhizobium japonicum bacteroids was found about the time of maximum nitrogenase inhibition. However, the accumulation of amino acids in soybean nodules lagged behind the inhibition of nitrogenase. Furthermore, in studies of a second legume, Phaseolus vulgaris (L.) inoculated with two different strains of Rhizobium phaseoli, a high nitrate treatment inhibited nitrogenase but had no significant effect on amino acid composition of nodules. The possibility that nitrate may interfere with the supply of carbon substrates to bacteroids was examined by the analysis of organic acids in legume nodules supplied with nitrate. Nitrate had a small (10-20%) negative effect on the concentration of tricarboxylic acid cycle acids in P. vulgaris nodules. However, in G. max nodules, high nitrate treatment resulted in significant increases in the concentration of malate, succinate, fumarate, and citrate. Thus, carbon deprivation of bacteroids also seems unlikely as a cause of the inhibition of nitrogenase by nitrate. There was a transient increase in ammonium concentration in P. vulgaris nodules in response to high nitrate treatment. This effect was rapid relative to other effects of nitrate on nodule composition and was roughly coincident with the rapid decline in acetylene reduction activity.     

Nitrite and Nitric Oxide as Inhibitors of Nitrogenase from Soybean Bacteroids

Nitrite was able to strongly inhibit C₂H₂ reduction by nitrogenase from soybean bacteroids, whereas H₂ evolution was unaffected under the same conditions. NO inhibited both C₂H₂ reduction and H₂ evolution; during C₂H₂ reduction, sensitivity of nitrogenase to NO was higher than to NO₂⁻, and the K_i values were, respectively, 0.056 and 0.52 mM. Production of NO resulting from a reduction of NO₂⁻ by dithionite in nitrogenase incubations was observed. However, the characteristics of inhibitions and the low level of NO generated by nitrite reduction ruled out the suggestion concerning a direct role of NO to explain the inhibitory effect of NO₂⁻ on nitrogenase.     

Nitrogenase Activity Associated with Halodule wrightii Roots

Nitrogen fixation (acetylene reduction) associated with roots of the seagrass Halodule wrightii was measured offshore near Beaufort and Moorhead City, N.C. Rates of acetylene reduction were higher in aerobic than in anaerobic assays and were linear for up to 5 days. The temperature range for acetylene reduction was 15 to 35°C with a maximum activity at 35°C. Nitrogenase activity was shown to vary seasonally with highest activities occurring during warmer summer months (23 μg of N₂ fixed per m² per day). At in situ temperature, nitrogenase activities associated with surface-sterilized and non-surface-sterilized roots were similar. One morphological bacterial type was isolated from surface-sterilized roots and identified as Klebsiella pneumoniae type 4B.     

Nitrogenase from Rhodospirillum rubrum Relation between ‘switch-off’ effect and the membrane component. Hydrogen production and acetylene reduction with different nitrogenase component ratios

Nitrogenase activity of ‘membrane-free’ extracts, produced from nitrogenstarved Rhodospirillum rubrum to which 4 mM NH⁺₄ had been added is only about 10% of the activity in the control. The activity could be restored to 80% by including the membrane component, earlier found to activate R. rubrum nitrogenase, in the reaction mixture. The relation between this ‘switch-off/switch-on’ effect and the function of the membrane component is discussed.Hydrogen production catalyzed by R. rubrum nitrogenase is also dependent on activation by the membrane component. Hydrogen production is inhibited by acetylene but the degree of inhibition is dependent on the nitrogenase component ratio. The strongest inhibition is achieved at low MoFe protein/Fe protein ratios. The $\text{ATP}\text{2 e}{}^{\mathrm{?}}$ values are 4–5 at the component ratios giving the highest activity and increase at high MoFe protein/Fe protein ratios. CO inhibits acetylene reduction but has no effect on the hydrogen production.