The symbiosis between Rhizobium leguminosarum and Pisum sativum : regulation of the nitrogenase activity

Although infected cell O2 concentration (Oi) is known to limit respiration and nitrogenase activity in legume nodules, techniques have not been available to measure both processes simultaneously in an individual legume nodule. Consequently, details of the relationship between nitrogenase activity and Oi are not fully appreciated. For the present study, a probe was designed that allowed open circuit measurements of H2 evolution (nitrogenase activity) and CO2 evolution (respiration rate) in a single attached soybean nodule while simultaneously monitoring fractional oxygenation of leghemoglobin (and thereby Oi) with a nodule oximeter. Compared to measurements of whole nodulated roots, use of the probe led to inhibition of nitrogenase activity in the single nodules. During oximetry measurements, total nitrogenase activity (TNA; peak H2 evolution in Ar/O2) in the single nodules was 16% of that in whole nodulated roots and 48% of nodulated root activity when Oi was not being measured simultaneously. This inhibition did not affect the nodules' ability to regulate Oi, because exposure to Ar/O2 (80:20, v/v) caused nitrogenase activity and respiration rate to decline, and this decline was linearly correlated with a concurrent decrease in Oi. When the nodules were subsequently exposed to a linear increase in external pO2 from 20 to 100% O2 at 2.7% O2/min, fractional leghemoglobin oxygenation first increased gradually and then more rapidly, reaching saturation at a pO2 between 76 and 100% O2. Plots of nitrogenase activity and respiration rate against Oi showed that rates increased with Oi up to a value of 57 nM, with half-maximal rates being attained at Oi values between 10 and 14 nM O2. The maximum nitrogenase activity achieved during the increase in pO2 (potential nitrogenase activity) was 30 to 57% of that measured in intact nodulated roots, showing that O2 limitation of nitrogenase activity could account for a significant proportion of the inhibition of TNA associated with the use of the probe. However, some factor(s) in addition to O2 must have limited the activity of single nodules at both subsaturating and saturating Oi. At Oi values greater than about 57 nM, nitrogenase activity and nodule respiration were inhibited, but, because this inhibition has been shown previously to be readily reversible when the Oi was lowered, it was not attributed to direct O2 inactivation of the nitrogenase protein. These results indicate that maximum nitrogenase activity in legume nodules is supported by a narrow range of Oi values. Possible biochemical mechanisms are discussed for both O2 limitation of nitrogenase activity at low Oi and inhibition of nitrogenase activity at high Oi.

Gluconacetobacter diazotrophicus is an N(2)-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O(2) pressures (pO(2)) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO(2) (5 to 60 kPa). Nitrogenase activity was measured by H(2) evolution in N(2)-O(2) and in Ar-O(2), and respiration rate was measured by CO(2) evolution in N(2)-O(2). To validate the use of H(2) production as an assay for nitrogenase activity, a non-N(2)-fixing (Nif(-)) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup(+)) activity (0.016 +/- 0.009 micromol of H(2) 10(10) cells(-1) h(-1)) when incubated in an atmosphere enriched in H(2). However, Hup(+) activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO(2) tested. However, when the assay atmospheric pO(2) was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO(2) for nitrogenase activity was 0 to 20 kPa above the pO(2) at which the bacteria had been grown. As atmospheric pO(2) was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO(2) was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO(2) from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O(2), 80% of nitrogenase activity was recovered within 10 min, indicating a "switch-off/switch-on" O(2) protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N(2) at a wide range of atmospheric pO(2) and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO(2).

The relationship between the rates of nitrogenase, nitrate reductase, and glutamine synthetase activities, and plant ontogeny in rice (Oryza sativa L.), cultivar ;M9', grown in salt marsh sediment with and without nitrate treatment was studied. In both treatments, nitrogenase activity measured as the immediate linear rate of acetylene reduction by bacteria associated with the roots varied with plant age. In control plants, the nitrogenase activity developed during the vegetative stage, peaked during early reproductive growth and then declined. The application of 10 kilograms N per hectare as KNO(3) once every 2 weeks delayed the development of and decreased the nitrogenase activity. The nitrogenase activity in both treatments developed as leaf nitrate reductase activity declined. The per cent nitrogen of roots was negatively correlated with the rates of acetylene reduction during the life cycles of control and nitrate-treated plants. This suggests that the concentration of combined nitrogen in the plants controlled the development and rate of root-associated nitrogenase activity. During reproductive growth, no nitrate reductase activity was detected in the roots from either treatment. In control plants, the patterns of nitrogenase activity and glutamine synthetase activity in the roots were similar. Thus, rice roots have the potential to assimilate ammonia while fixing N(2). During the vegetative and early reproductive stages of growth, the development of maximal rates of nitrogenase activity coincided with an increase of total nitrogen of the plants in both treatments.

Biochar is produced from the pyrolysis of organic materials, and when buried in soil can act as a long term soil carbon (C) store. Evidence suggests that biochar can also increase crop yields, reduce nutrient leaching and increase biological nitrogen fixation in leguminous plants. However, the potential for increasing biological N2 fixation in agroecosystems is poorly understood, with inconsistent reports of root nodulation following biochar application. Therefore, the aim of this study was to determine the effect of biochar application rate and time since application on nodulation and nitrogenase activity in nodules of clover grown in a temperate agricultural soil. We used replicated field plots with three biochar application rates (0, 25 and 50 t ha−1). Three years after biochar amendment, the plots were further split and fresh biochar added at two different rates (25 and 50 t ha−1) resulting in double-loaded reapplications of 25 + 25 and 50 + 50 t ha−1. Three years after biochar application, there was no significant difference in the total number of root nodules between biochar-amended and unamended soil, regardless of the application rate. However, despite clover root nodules being of a similar number and size the level of nitrogenase activity of individual nodules in biochar-amended soil was significantly higher than in unamended soil. Reapplication of biochar resulted in decreased nodulation, although the rate of nitrogenase activity per nodule remained unaffected. In the short term, biochar influences root nodule number and localised N2 fixation per nodule; however, total nitrogenase activity for the whole root system remained unaffected by the application rate of biochar or time since its application. These results emphasise the importance of long-term field studies, with a variety of applications rates for determining the influence of biochar applications on N2-fixing organisms and in providing data that can meaningfully inform agronomic management decisions and climate change mitigation strategies.

Nodules of Rhizobium leguminosarum bv. phaseoli in symbiosis with Phaseolus vulgaris were compared with regard to their nitrogenase activity and activities of enzymes involved in the removal of O2·- and H2O2 as well as total ascorbate content. Activities of catalase (EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11), and total ascorbate content were consist­ently higher in nodules inhabited by bacterial strains with higher nitrogenase activity. Values for superoxide dismutase (EC 1.15.11), and guaiacol peroxidase activity did not differ for the bacterial strains compared. On the other hand, when different plant cultivars were inoculated with the same bacterial strain, high nitrogenase activity did not correlate with a higher activ­ity of the oxygen scavenging enzyms or a higher content of total ascorbate. In this case, values for guaiacol peroxidase activity were greatly enhanced in nodules with lower nitrogen­ ase activity. This may be part of a hypersensitive reaction of the plant cultivar against the bacterial symbiotic partner. Inhibition of catalase activity in the nodules by addition of triazole to the nutrient solution did not alter nitrogenase activity within the first nine hours after addition. It can be concluded that the activity of catalase, ascorbate peroxidase, and superoxide dismutase is not generally coupled to nitrogenase activity in root nodules of P. vulgaris.

Two aspects of the metabolism in pea root nodules in relation to nitrogen fixation have been studied. The role of malate dehydrogenase in the carbon metabolism and the efficiency of the nitrogenase reaction in intact root nodules was investigated. The presence of a root nodule stimulated form of malate dehydrogenase is demonstrated. From a comparison of the kinetic properties of this enzyme and the enzyme from root cells, it is concluded that the root nodule form of malate dehydrogenase is capable of allowing a high flux of malate production from oxaloacetate and also to establish a sufficient oxaloacetate concentration necessary for the synthesis of amino acids involved in the transport of newly fixed nitrogen from the root nodules. Data are presented to demonstrate that the variable H2 production by nitrogenase in intact root nodules is not caused by O2 limitation of the bacteroids but by plant factors like free fatty acids.

Root nodule development, and seasonal patterns of nodular nitrogenase and hydrogenase activities were determined for 5- to 8-year old black alder (Alnus glutinosa (L.) Gaertn.) and Russian olive (Elaeagnus angustifolia L.) interplanted with black walnut (Juglans nigra L.) on bottomland and upland sites in central Illinois, USA. Black alder produced nodules at both sites, but Russian olive did so only at the bottomland site. Nodular nitrogenase activity was detectable in both species over a 220-day period. Maximum, midday rates of nitrogenase activity (acetylene reduction) of 15 to 20 micromoles C(2)H(4) per g dry nodule per hour were maintained by black alder for approximately 150 days at both the upland and bottomland sites. Near maximum rates of nodular nitrogenase activity were maintained for a similar period by Russian olive at the lowland site, although specific nitrogenase activity was approximately 25% lower than in black alder owing to a larger proportion of necrotic nodular tissue in Russian olive. In both species, nitrogenase activity increased exponentially with temperature between 10 degrees C and 20 to 25 degrees C. No net hydrogen evolution by nodules of either species was detected at any time during the assay period, indicating efficient hydrogenase systems were operating under the conditions of the field assay. Height of black walnut interplanted with nodulated black alder and Russian olive was greater than that of black walnut grown in pure stands.

Effect of cropping systems and arbuscular mycorrhizal fungi on soil microbial activity and root nodule nitrogenase

Inhibition of nitrogenase (EC 1.18.6.1) activity by O2 has been suggested to be an early response to disturbance in carbon supply to root nodules in the Frankia‐Alnus incana symbiosis. Intact nodulated root systems of plants kept in prolonged darkness of 22 h were used to test responses to O2 and short‐term N2 deprivation (1 h in Ar:O2). By using a Frankia lacking uptake hydrogenase it was possible to follow nitrogenase activity over time as H2 evolution in a gas exchange system. Respiration was simultaneously recorded as CO2 evolution. Dark‐treated plants had lower initial nitrogenase activity in N2:O2 (68% of controls), which declined further during a 1‐h period in the assay system in N2:O2 at 21 and 17% O2, but not at 13% O2. When dark‐treated plants were deprived of N2 at 21 and 17% O2 nitrogenase activity declined rapidly to 61 and 74%, respectively, after 20 min, compared with control plants continuously kept in their normal light regime. In contrast, there was no decline in dark‐treated plants at 13% O2, and only a smaller and temporary decline in control plants at 21% O2. When dark‐treated plants were kept at 21% O2 during 45 min prior to N2 deprivation at 17% O2 the decline was abolished. This supports the idea that the decline in nitrogenase activity observed in N2:O2 at 21% O2 and during N2 deprivation was caused by O2, which affected a sensitive nodule fraction. Nodule contents of the amino acids Gln and Cit decreased during N2 deprivation, suggesting decreased assimilation of NH4+. Contents of ATP and ADP in nodules were not affected by short‐term N2 deprivation. ATP/ADP ratios were about 5 indicating a highly aerobic metabolism in the root nodule. We conclude that nitrogenase activity of Alnus plants exposed to prolonged darkness becomes more sensitive to inactivation by O2. It seemed that dark‐treated plants could not adjust their nodule metabolism at higher perceived pO2 and during cessation of NH4+ production.

SUMMARY: The effects of purified oxyleghaemoglobin added to suspensions of bacteroids prepared anaerobically from soybean root nodules, were studied in terms of uptake of dissolved O2, nitrogenase activity and dissolved O2 concentration, in the absence of a gas phase. Leghaemoglobin allowed maximum rates of O2 uptake to continue to a much lower range of concentrations of free dissolved O2 than in the absence of the protein. This effect was diminished when the leghaemoglobin concentration was less than about 50 μ m. Nitrogenase activity at a given O2 concentration was not increased by raising the leghaemoglobin concentration above about 50 μ m. The fractional oxygenation of leghaemoglobin giving half the maximal O2 consumption rate by bacteroids was usually about 0·2; with limiting leghaemoglobin concentrations it was higher. Rates of nitrogenase activity were invariably greater during periods when the discharge of O2 from oxyleghaemoglobin was occurring at the maximum rate, than when similar maximum O2 uptake rates were being supported by higher concentrations of free dissolved O2. When myoglobin was used as the O2 carrier protein in similar experiments, enhanced nitrogenase activity also accompanied the maximum rate of discharge from the carrier. This occurred at about four times the concentration of free dissolved O2 at which it occurred when leghaemoglobin was the carrier. These results are discussed in relation to current theories about the mechanism of leghaemoglobin action.

Six experiments, two each in the phytotron, greenhouse, and field, were conducted to assess the contribution of nitrogenase activity (acetylene reduction) by stem nodules in the presence and absence of root nodules of Sesbania rostrata (Brem & Oberm). In a greenhouse experiment, the effect of detaching already formed aerial stem nodules on the restoration of root nodules and nitrogenase activity was studied. The field experiment compared nodulation and acetylene-reduction activity by dual-nodulating S. rostrata and root-nodulating Sesbania cannabina. Acetylene-reduction activity expressed per gram of nodule dry weight was higher for stem nodules than for root nodules. Root nodule dry weight and acetylene-reduction activity failed to increase after stem inoculation, but root nodule dry weight and acetylene-reduction activity increased several fold within 15 days of detachment of aerial stem nodules. Stem nodulation, which occurred without inoculation under lowland field condition, suppressed root nodulation, thus accounting for more than 75% of total nitrogenase activity. Sesbania rostrata showed higher acetylene-reduction activity than S. cannabina. In dual-nodulating plants, root and stem nodules appeared to strike a balance in competition for energy, which may be controlled by stem nodulation. Key words: Sesbania rostrata, Azorhizobium caulinodans, stem nodule, root nodule, acetylene-reducing activity.

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.

Plants ofAlnus incana (L.) Moench in symbiosis with a local source ofFrankia were exposed to prolonged darkness under controlled climate conditions.Frankia vesicle clusters were prepared from the root nodules, and the condition ofFrankia was measured as respiratory capacity by supplying the preparation with saturating amounts of four different substrates. During darkness, nitrogenase (EC 1.7.99.2) activity decreased in intact plants and in the vesicle-cluster preparations. The respiratory capacity ofFrankia also decreased. After 4 d in darkness most respiration was lost, though all nitrogenase activity was already lost after 3 d. When the dark treatment was ended after 2 d and normal light/dark conditions restored, nitrogenase activity immediately started to recover. The respiratory capacity continued to decrease and no recovery was observed until the third day after the end of the dark treatment. Whole-plant nitrogenase activity slowly increased at a rate similar to the rate of increase observed in untreated plants. Transmission electron micrographs of the root nodules showed that the cytoplasm of infected host cells and the cells ofFrankia were structurally degraded in response to dark treatment, while young vesicles were frequent during recovery. Growth and differentiation ofFrankia cells were apparently important for recovery of the enzyme activities studied.

Isotopic15N2 experiments confirmed nitrogen fixation inParasponia parviflora. The conversion ratio C2H4/N2 was 6.7 under the experimental conditions employed. Measurements of the δ15N in leaves of Parasponia and Trema showed on the basis of these determinations thatParasponia parviflora possesses N2-fixing capacity and can be distinguished in this respect from the non-nitrogen-fixingTrema cannabina tested by the same method. Therefore, δ15N can be used to monitor N2 fixation in natural ecosystems. Hydrogen evolution and the relative efficiency of N2 fixation in this relation have been determined. DetachedParasponia parviflora root nodules grown in soil and tested in an argon/oxygen atmosphere produced appr. 4 μmol H2.h−1.g−1 fresh weight root nodules. The relative efficiency of hydrogen utilization as measured in argon, air, and in the presence of C2H2 10% (v/v) was for both equations\(\left( {1 - \frac{{H_2 (air)}}{{H_2 (Ar)}}} \right) and \left( {1 - \frac{{H_2 (air)}}{{C_2 H_2 }}} \right)\) used for to express this efficiency 0.96 and 0.97, respectively. This indicates that Parasponia like the root nodules of some actinorhizal symbioses (Alnus, Myrica, Elaeagnus) and some tropical legumes (Vigna sinensis) has evolved mechanisms of minimizing net hydrogen production in air, thus increasing the efficiency of electron transfer to nitrogen. The oxygen relation of nitrogen fixation (C2H2) inParasponia parviflora root nodules was determined. The nitrogenase activity of Parasponia root nodules increased at increasing oxygen concentrations up till c. 40% O2. At oxygen levels above 40% O2, the nitrogenase activity of the root nodules was nil or very erratic suggesting that at these oxygen levels the nitrogenase is not longer protected against the harmful effect of oxygen. In this respect Parasponia root nodules differ from actinorhizal root nodules in other nonlegumes, where optimal nitrogenase activity was observed in the range of 12–25% oxygen. Respiration experiments with Parasponia root nodules showed that in the range 10, 20, and 40% oxygen, the respiration rate (CO2 evolution) increased concomitantly with an increase of the acetylene reduction rate. The CO2/C2H4 values obtained varied between 8.1 and 19.2, being therefore 2–3 times higher than similar estimations in some actinorhizal and legume root nodules. The respiratory quotient (RQ) of detachedParasponia parviflora root nodules was in air initially approximately 2.0, but this value dropped to about 1.0 in a 3-hours period.

Response of respiration and nitrogenase activity in <i>Datisca glomerata</i> (Presl.) Baill. to changes in pO₂