Occurrence and activity of hydrogenase in symbiotic Frankia from field-collected Alnus incana

Occurrence and activity of the hydrogen uptake enzyme were studied in root nodule homogenates made from plants of Alnus incana (L.) Moench collected from field sites in the northern part of Sweden. Nitrogenase (EC 1.7.99.2) activity (estimated by acetylene reduction) and hydrogen evolution were studied in excised nodules. All Frankia sources showed acetylene reduction activity, and possessed a hydrogen uptake system. Hydrogen uptake in nodule homogenates from the Frankia sources measured at 23.8 μM H₂ ranged from 0.04 to 5.0 μmol H₂ (g fresh weight nodule)⁻¹ h⁻¹. The H₂ uptake capacity of nodule homogenates from one of the Frankia sources was almost 8 times higher than the hydrogen evolution from nitrogenase, both expressed on a nodule fresh weight basis. Frankia sources from field sites 6 and 11 showed K_m for H₂ of 13.0 and 23.6 μM H₂, respectively. This indicates similarities in the hydrogen uptake enzymes in the two Frankia sources. It is concluded that hydrogen uptake is a common characteristic in Frankia.     

Effects of elevated oxygen tensions on acetylene reduction in Alnus incana-Frankia symbioses

An open flow-through gas system was used to determine the effect of C₂H₂ and elevated O₂ on acetylene reduction activity (ARA) and respiration of the intact, potted root system of Alnus incana (L.) Moench in symbiosis with Frankia Avcll or with a local source of Frankia . Both symbiotic systems responded to C₂H₂ by an immediate plateau range in ARA. The Plateau in ARA was in some cases followed by a decline of less extent than reported for many legumes. A concurrent decline in net respiration of the root system was on average 8% of the CO₂ efflux prior to C₂H₂ introduction.
Respiration of the root systems in both symbioses responded to elevated oxygen levels in the 10 kPa C₂H₂ atmosphere by an increase of up to 17% of the net respiration prior to C₂H₂ introduction in 21 kPa O₂. In contrast, the elevated oxygen levels resulted in an immediate drop in ARA followed by a minor increase to a stable level lower than that at the preceding, lower oxygen tension. The symbiosis with the local Frankia had lost all ARA when the partial pressure of O₂ exceeded 50 kPa, whereas the symbiosis with Avcll still had some activity at 80 kPa O₂. This difference in tolerance of elevated O₂ clearly shows that the oxygen exclusion mechanisms may be controlled by the microsymbiont in Alnus-Frankia symbioses. The symbiotic systems recovered ARA to a similar extent when returned from elevated O₂ levels to 21 kPa O₂.     

Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina

Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated. Acetylene reduction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickel-depleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 μ mol H₂ (mg protein)⁻¹ h⁻¹ after addition of up to 1.0 μ M Ni. It is likely that the hydrogenase activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia living in symbiosis with Casuarina spp. show hydrogenase activity. Therefore, the results also indicate that the hydrogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina ., i.e., Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.     

Hydrogen metabolism of Casuarina root nodules: A comparison of two inoculum sources

Hydrogen metabolism was studied in three Casuarina species, C, equisetifolia Forst., C. glauca Sieb. ex. Spreng. and C. obesa Miq., either inoculated with the pure Frankia culture HFP CcI3 or inoculated with a crushed nodule inoculum made from C. glauca nodules. Nitrogenase (EC 1.7.99.2) activity and hydrogen evolution was measured on intact plants, while hydrogen uptake was measured on excised nodules and in nodule homogenates.
Nitrogenase activity was highest in C. glauca inoculated with C. glauca nodules, while no hydrogen evolution was detected. Hydrogen evolution was highest in the symbiosis between C. equisetifolia and HFP CcI3, but the nitrogenase activity showed intermediate values compared to the other symbioses. Measured at a concentration of 93 μ M H₂, H₂ uptake was highest in C. glauca inoculated with the C. glauca inoculum. H₂ uptake activity in homogenates was 83% of the intact nodule rate. With phenazinemethosulfate as the electron acceptor, H₂ uptake by nodule homogenates showed typical Michaelis-Menten kinetics with a K_m of 21.3 μ M for H₂.
The data presented here indicate a host plant effect on the endobiont which alters the hydrogen metabolism.     

Nitrogen fixation and biomass production in symbioses between Alnus incana and Frankia strains with different hydrogen metabolism

Three different strains of Frankia , the pure cultures AvcI1 and CpI1 and a local strain (crushed nodule inoculum), were compared in symbiosis with one clone of Alnus incana (L.) Moench. Hydrogen metabolism, nitrogenase (EC 1.7.99.2) activity and relative efficiency of nitrogenase were studied as well as growth and nitrogen content of the plants. The local Frankia strain showed no measurable hydrogen uptake but high H₂-evolution. No H₂-evolution was detected in Frankia AvcI1 because of its hydrogenase activity. CpI1 also had hydrogenase, although only a very small H₂-evolution was detected at the end of the growth period. Hydrogenase activity was detected both in pure cultures and nodule homogenates of CpI1 and AvcI1. Growth, biomass production and nitrogen content were highest in alders inoculated with Frankia AvcI1 while the lowest values were found for alders living in symbiosis with the local Frankia strain. The presence of hydrogenase in Frankia seemed to be benefical for growth and biomass production in the alders. However, the strains also differed with respect to spore formation. The local strain, but not AvcI1 and CpI1, formed spores in the root nodules.     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.     

Effect of H2 evolution on 15N2 fixation, C2H2 reduction and relative efficiency of leguminous symbionts

The (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratios of 15 clover- Rhizobium symbionts. soybean, and black medick symbionts were measured. Relative efficiency based on the C2H4 production and on ¹⁵N₂ incorporation were compared, and in most symbionts there was little difference between the two measures of relative efficiency. Total measurable electron flux through nitrogenase during acetylene reduction and ¹⁵N₂ incorporation were nearly equal for most symbionts studied. The relative efficiency and the (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratio showed an inverse correlation. Use of this ratio appears preferable to use of the ratio of C2H2 reduction/N₂ reduction. Some evolution of H₂ was observed in the presence of C₂H₂.     

Superoxide dismutase, catalase and nitrogenase activities of symbiotic Frankia (Alnus incana) in response to different oxygen tensions

Presence and activity of the enzymes superoxide dismutase (SOD) and catalase were studied in Frankia in symbiosis with Alnus incana (L.) Moench. Analysis on native PAGE gels indicated that symbiotic Frankia contained an FeSOD and catalase. The activity of the enzymes was in the same range as reported for cultured Frankia . Attempts to characterize SOD by western blots with antisera from Escherichia coli and Azotobacter vinelandii did not give clear-cut results with the antibodies used. Alnus incana plants were grown with the root system in 5, 10, 21 or 40% O₂ for up to 6 days. Nitrogenase activity, measured as ARA (acetylene reducing activity) dropped within 3 h when roots were exposed to low or high oxygen. At 40% O₂ ARA was almost completely lost while at 5 and 10% O₂ ARA decreased to 69 and 74% of the inital value, respectively, Nitrogenase activity recovered at ail oxygen tensions. Recovery rates resembled the continuous increase in ARA in plants continuosly kept at 21% O₂, and suggests that new vesicles with envelopes of appropriate thickness were formed. The ARA measurements confirm results from an earlier study where nitrogenase activity was measured as H₂ evolution. There was a tendency for increased SOD and catalase activities in Frankia from root systems exposed to 40% O₂ for 24 h but not earlier or later than this. When data from all experimental times were pooled. SOD activity increased significantly with increased oxygen tension whereas catalase activity decreased. Although ARA per plant varied with oxygen tension, there was no statistically significant correlation between ARA and SOD or between ARA and catalase. It seems that being linked to nitrogenase activity is only one role of SOD and catalase in this symbiotic Frankia .     

Interaction between hydrogenase, nitrogenase, and respiratory activities in a Frankia isolate from Alnus rubra

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in NH4+-grown, undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation.     

Reduction of nitrous oxide by a soil Cytophaga in the presence of acetylene and sulfide

Abstract A denitrifying Cytophaga was isolated from soil enriched by anaerobic incubation with glucose, sulfide (S²⁻), nitrous oxide (N₂O), and acetylene (C₂H₂). Such soil enrichments and pure cultures of the isolated Cytophaga reduced N₂O rapidly even in the presence of a normally inhibitory concentration of C₂H₂ (4 kPa) providing S²⁻ was present (8 μmol/g soil or 0.4 μmol/ml culture). Since C₂H₂ inhibition of the reduction of N₂O is used as a tool in the assay of denitrification, the presence in large numbers of such a Cytophaga may influence the effectiveness of this assay especially in sulfidic environments.     

Appearance of a reversible hydrogenase activity in Anabaena cylindrica grown in high light

In vivo H₂ evolution by Anabaena cylindrica Lemm. strain PCC 7122 grown in the presence of ammonia at low and high light intensities was studied. We found that after 2 h of anaerobic incubation, H₂ evolution [at a rate of 0.5 μmol (mg dry weight)¹ h⁻¹] via reversible hydrogenase occurred in high light grown cells, while this kind of activity was not found in low light grown cells. H₂ evolution was inhibited by 3-(3'. 4'-dichlorophenyl-1, 1-dimethylurea (DCMU). Illuminating the cells in the phycocyanin absorption region resulted in a higher rate of H₂ evolution than illuminating the cells in the chlorophyll absorption region. The results indicate that reversible hydrogenase receives reducing equivalents from photosynthetic water photolysis and that both photosystem II and photosystem I participate in the H₂ production. Hydrogenase activity was found in the soluble fraction after mild sonication in the case of low light grown cells. After this treatment high light grown cells retained 70% of their hydrogenase activity in the particulate fraction, but released it into the soluble fraction in the presence of 2% deoxycholic acid.
In vitro H₂ evolution did not differ significantly in the low and high light grown cells. Hence, the differences in the in vivo H₂ evolution reflect the different availability of endogenous reductants for hydrogenase in the two kinds of cells. On the basis of our results it is suggested that high light grown Anabaena cells eliminate part of the photosynthetically produced excess electrons via an induced reversible hydrogenase activity. This is the first report of H₂ evolution associated with water photolysis and catalyzed by hydrogenase in cyanobacteria.     

Uptake hydrogenase system in urd bean (Vigna mungo) Rhizobium in relation to nitrogen fixation

Twenty-five isolates of urd bean ( Vigna mungo ) Rhizobium were tested for the presence of an H₂-uptake system using triphenyl tetrazolium chloride reduction as the screening procedure. The isolates which reduced the dye rapidly at early stages of growth were found to recycle H₂ both in culture as well as in nodules. H₂-uptake positive, H₂-uptake negative strains and H₂-uptake negative mutants were compared on the basis of their effect on dry matter accumulation and N₂-fixation. Greenhouse experiments have shown the beneficial effect of the H₂-uptake positive system in nodules on N₂-fixation. Plants inoculated with H₂-uptake positive strains produced higher N content and dry matter over the plants inoculated with H₂-uptake negative strains.     

Photosynthetic oxygen evolution within Sesbania rostrata stem nodules

The tropical wetland legume, Sesbania rostrata Brem. forms N₂-fixing nodules along its stem and on its roots after infection by Azorhizobium caulinodans . The N₂-fixing tissue is surrounded by a cortex of uninfected cells which, in the stem nodules (but not the root nodules), contain chloroplasts. The photosynthetic competence of these chloroplasts was assessed through a novel technique involving image analysis of chlorophyll a fluorescence. Calculation of the quantum efficiency of photosystem II (PS II) photochemistry from these images indicated that most of the chloroplasts with potential for non-cyclic photosynthetic electron transport were concentrated within the mid- and inner-cortex, close to the edge of the N₂-fixing tissue. PS II activity in the cortical cells was confirmed in vivo using O₂-specific microelectrodes which showed that the concentration of O₂ (pO₂) in the outer cortex could rise from less than 1% up to 23.4% upon increased irradiance of the nodule, but that the pO₂ of the inner cortex and infected tissue remained less than 0.0025%. Nitrogenase activity of stem nodules, as measured using a flow-through acetylene reduction assay (no H₂ evolution was evident), showed a reversible increase of 28% upon exposure of the nodules to supplemental light. This increase resembled that obtained with stem nodules upon their exposure to an external pO₂ of 40%.     

C2H2 and Ar induce rapid declines in nitrogenase activity and CO2 evolution in nodules of Datisca glomerata

Preliminary studies have indicated that after addition of C₂H₂ there is a rapid decline in nitrogenase activity in the nodules of Datisca glomerata . The present work was undertaken to determine whether (1) there is also a decline in respiration and (2) the decline is associated with the cessation of ammonia production. The rates of C₂H₄ and CO₂ evolution by nodulated root systems of Datisca were measured as a function of time after exposure to C₂H₂. The peak rate of C₂H₄ evolution occurred at 30 s after C₂H₂ exposure, while the rate of CO₂ evolution started to decline at 60 s after exposure to C₂H₂. Incubation of nodules in a gas mixture containing Ar also caused a decline in CO₂ evolution. Further, pretreatment with Ar eliminated most of the C₂H₂-induced decline in nitrogenase activity and CO₂ evolution. These C₂H₂- and Ar-induced declines in Datisca nodules are more rapid than those reported in any other nodules. They are evidence that continued ammonia formation is essential for maintenance of normal nitrogenase activity in Datisca nodules.     

Nitrogenase activity decay and energy supply in Frankia after addition of ammonium to the host plant Alnus incana

A clone of Alnus incana (L.) Moench was grown in symbiosis with a local source of Frankia or with Frankia Ar14. Seven to 9-week-old plants were given 20 m M NH₄Cl (20 m M KCl = control) for 3 days. Nitrogenase activity of intact plants decreased gradually within the 3 days of treatment to about 10% of the initial rates. Hydrogen evolution in air and total nitrogenase activity responded similarly to the treatment. Relative efficiency of nitrogenase thus remained the same throughout the study period. Control plants were not affected. Measurements of nitrogenase activity in root nodule homogenates (in vitro measurements) indicated loss of active nitrogenase rather than shortage of energy for nitrogenase activity in Frankia from ammonium-treated plants. Shoots were exposed to ¹⁴CO₂ and translocation of ¹⁴C to Frankia vesicle clusters prepared from root nodules was studied. Frankia vesicle clusters from ammonium-treated plants contained about half as much ¹⁴C as those of control plants during all 3 days studied. One explanation for the observed effects is that a reduced supply of carbon to Frankia vesicles in the root nodules caused a reduced metabolic rate, including reduced protein synthesis and synthesis of nitrogenase.     

Photoproduction of hydrogen, nitrogenase and hydrogenase activities of free and immobilized whole cells of Rhodobacter sphaeroides O.U. 001

Abstract Photoproduction of hydrogen, nitrogenase activity (acetylene reduction) and hydrogenase activity (methylene blue dye reduction) were studied in free and alginate immobilized whole cells of a purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides O.U. 001. Four-fold increase in hydrogen production, two-fold increase in nitrogenase activity and 1.2-fold increase in the hydrogenase activity were observed in immobilized cells compared to free cells. Effect of various inhibitors (CO and C₂H₂) and electron donor (H₂) on the above three functions by free and immobilized cells has also been studied.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

The effectivity of Frankia for nodulation and nitrogen fixation in Alnus rubra and A. glutinosa

The effectivity of nodulation of Alnus rubra Bong, by Frankia isolates from A. rubra and Alnus glutinosa (L.) Gaertn. in Northern Britain was compared with strains from The Netherlands and North America, using plants grown in combined nitrogen-free conditions. All strains gave rise to spore (-) nodules, even when isolated from nodules from sites known to contain spore (+) nodules. Nodules of all plants evolved little hydrogen, probably due to the presence of an efficient uptake hydrogenase in the microsymbkmts. Nodule weight as a percentage of whole plant weight was higher for nodules of low specific activity (N fixed per unit weight nodules), attaining a maximum of 5.1% of plant dry weight in the least effective of the heterologous associations of A. glutinosa Frankia with A. rubra . The range of variation in nodule specific activity was much greater in heterologous than homologous associations, but nodules of high specific activity were found in both associations. However, plants that fixed most N during the growth period were not those with nodules of highest specific activity. The most effective associations were homologous symbioses, which combined good nodule growth per plant with satisfactory specific activity, fixing N at rates which would support superior plant growth under the prevailing growth conditions. Preliminary field experiments suggest that the most effective of the A. rubra isolates is suitable for use as an inoculant in nurseries. Strains isolated from A. glutinosa were more effective and showed a different order of effectivity in homologous symbioses compared with their association with A. rubra . An A. glutinosa strain was isolated, which stimulated satisfactory nodule growth and gave good nodule specific activity in both A. rubra and A. gtutinosa .     

The Glycine-Glomus-Bradyrhizobiun symbiosis. XI. Nodule gas exchange and efficiency as a function of soil and root water status in mycorrhizal soybean

Soybean [ Glycine max (L.) Merr. cv. Hobbit] plants were inoculated with a HUP− strain of Bradyrhizobium japonicum (Nitragin 61A118) and either colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol & Gerd.) Gerd. and Trappe or fertilized with KH₂PO₄ (nonVAM). They were grown for 50 days in a growth chamber and harvested over a 4-day drought period during which available soil water decreased to 0. Nodule P concentrations and P-use efficiency declined linearly with soil and root water content during the harvest period in both VAM and nonVAM plants. Nitrogenase activity, estimated from H₂ evolution and C₂H₂ reduction data, was also a linear function of declining nodule P concentrations and CO₂-exchange rates and showed simular patterns in both treatments. Hydrogen evolution and the relative efficiency of N₂ fixation, on the other hand, reacted differently to increasing drought in VAM and nonVAM plants. Differences in the responses of nodule activity in VAM and nonVAM plants to drought are interpreted in terms of demand for nodule P and carbohydrates and of the effects of dehydration on O₂ diffusion through nodule tissue.     

Effect of low rhizosphere oxygen on growth, nitrogen fixation and nodule morphology in lucerne

Oxygen shortage in soils can occur following a wide range of natural circumstances, affecting the plant's physiology. In this paper the performance of nodulated lucerne plants under severe hypoxia is examined and the mechanisms involved to achieve this adaptation are discussed. Nodulated lucerne plants ( Medicago sativa L.) were grown with their rooting medium exposed to 1 or 21 kPa oxygen. Final yield, as expressed on a shoot dry weight basis, was unaffected but root and nodule dry weights were reduced by 50%. Water content in roots and nodules was higher at 1 kPa as a result of the formation of aerenchyma. Specific acetylene reduction activity was higher in hypoxic nodules as a consequence of modified nodule structure, although they were more sensitive to the presence of acetylene or nitrate. Root respiration was insensitive to changes in external oxygen supply, therefore providing adequate support for mineral uptake. Nodule respiration rates were 5 times higher in control plants when measured as CO₂ evolution, whereas no differences were observed in O₂ uptake. It is suggested that adaptation of nodulated lucerne to low oxygen concentrations involves changes in photosynthate allocation and nodule morphology, which provide a more efficient nitrogen fixation.