Potential Nitrogenase Activity Research Articles

Biological activity of sod-podzolic soils at different levels of radioactive contamination

The biological (enzymatic) activity of sod-podzolic soils has been investigated in the zone of mandatory resettlement and in the alienation zone of the Chornobyl Nuclear Power Plant (CNPP) under the influence of ionizing radiation. Gas chromatographic methods were used to determine potential nitrogenfixing (nitrogenase) activity, biochemical methods were applied to study the activity of hydrolytic enzymes (cellulase and protease) responsible for the decomposition of plant residues in the soil, as well as enzymes involved in oxidative-reductive reactions of organic matter transformation (catalase and polyphenoloxidases). Statistical analysis was also employed. The results of the conducted research indicate that a relatively low increase in the total dose rate in the radioactive contamination area (from 0.2 to 1.6 µGy/h) at Polygon No. 1 (the zone of mandatory resettlement near the village of Khrystynivka, Narodychi district, Zhytomyr region) stimulated potential nitrogenase activity of soil diazotrophs and the activity of both hydrolytic enzymes and oxidoreductases. High dose rates (up to 22.2, 61.6, and especially up to 84.0 mGy/h) at Polygon No. 2, located in the alienation zone of the CNPP directly in the area of the former Red Forest, led to a decrease in soil enzymatic activity. Thus, the obtained results unequivocally demonstrate the different nature of the impact of ionizing radiation levels on the biological activity of sod-podzolic soils. The more radioactive soil in the zone of mandatory resettlement stimulates the enzymatic activity of soil microbiota. This could be attributed to adaptive changes in the development and activity of microorganisms or changes in the composition of their communities, with a prevalence of radio-tolerant microbiota representatives. It is also possible that the manifestation of a radiobiological effect, such as radiation stimulation (radiation hormesis), accelerating the growth and development of microorganisms, may occur. In the alienation zone of the CNPP, with high radioactive contamination, the negative influence of ionizing radiation on the metabolism of soil microorganisms is preserved. The obtained results are highly correlated with the indicators of the abundance of nitrogen-fixing microorganisms in the investigated soils, as well as microorganisms representing the saccharolytic (fungi and cellulolytic bacteria) and peptolytic (ammonifiers) pathways of plant residue degradation, as previously demonstrated by us.

Potential mechanisms of soil nitrogen content heterogeneity associated with biocrust development in drylands

AbstractBiological soil crusts (biocrusts) are complex communities of micro‐ and macro‐organisms dwelling at the soil surface in dryland regions worldwide, which contribute to important ecological functions in these areas. Biocrusts may reach different developmental stages, associated to autogenic succession of specific phototrophic organisms. However, notwithstanding the large amount of existing literature, little is known regarding the relationship between biocrust stages of development and soil nutrient dynamics. In this opinion paper, we specifically focused on soil nitrogen, and compared the total nitrogen content and potential nitrogenase activity of different developmental types of biocrust (‐covered) soils. Based on published reports, we looked at a possible relationship between nitrogen content at the soil surface and biocrust development, discussing the potential mechanisms leading to the observed soil nitrogen content heterogeneity. The results showed that a higher nitrogen content remained associated to the presence of moss‐dominated biocrusts, and this biocrust morphotype was characterized by a lower potential nitrogenase activity compared to the cyanobacteria‐ and/or lichen‐dominated biocrusts. We hypothesized that these seemingly contradictory findings might be attributed to three potential mechanisms: nitrogen inheritance, nutrient retention and nutrient transfer, which we hereby examined one by one. Altogether, our opinion supports the theory of biocrust succession from incipient cyanobacteria‐dominated stages to more ‘mature’ stages dominated by mosses. We stress how the heterogeneous distribution of soil nitrogen, which is closely related with biocrust development and community types, eventually affects regional and even global nitrogen dynamic and storage.

Adaptations of Potential Nitrogenase Activity and Microbiota with Long-Term Application of Manure Compost to Paddy Soil

Biological nitrogen fixation complements nitrogen from fertilizers in crop plants under natural conditions. It also contributes to the reduction in chemical fertilizer (CF) utilization in cultivated lands, which fits the concept of sustainable agriculture. From this viewpoint, however, it is still unknown in paddy fields how soil bacterial nitrogenase and microbiota are affected by applied materials in the soil. Therefore, in this study, the effects of long-term material applications on potential nitrogenase activity and the microbiota of soil bacteria were investigated. The nitrogenase activity tended to be higher in manure compost (MC)-applied soils than in CF-applied soils in both summer and winter. Soil bacterial alpha diversity increased whereas soil ammonia availability decreased with the MC application. The dynamic response of soil bacterial microbiota was caused by the MC application. The abundance of Nitrospira, a class of ammonia and nitrite oxidation bacteria, was lower and the abundance of alpha-Proteobacteria was higher in the MC-applied soils than in the CF-applied soils. These results suggest that the alpha diversity increase and restricted availability of NH3-N might contribute to the increase in potential nitrogenase activity in the long-term MC-applied soils.

Composition and Activity of N2-Fixing Microorganisms in Mangrove Forest Soils

Mangrove forests are considered to be a highly productive ecosystem, but they are also generally nitrogen (N)-limited. Thus, soil N2 fixation can be important for the stability of both mangrove ecosystem functions and upland N supply. This study evaluates the N2 fixation activity and composition of relevant microorganisms in two coastal mangrove forests—the Guandu mangrove in an upstream estuary and the Bali mangrove in a downstream estuary—using the acetylene reduction method, real-time polymerase chain reaction, and next-generation sequencing. The results demonstrated that ambient nitrogenase activity was higher in downstream mangrove forests (13.2–15.6 nmol h−1 g−1 soil) than in upstream mangrove forests (0.2–1.4 nmol h−1 g−1 soil). However, both the maximum potential nitrogenase activity and nitrogenase gene (nifH gene) copy number were found to be higher in the upstream than in the downstream mangrove forests, implying that the nitrogenase activity and diazotrophic abundance may not necessarily be positively correlated. In addition, amended MoO4 (which inhibits the activity of sulfate-reducing bacteria in N2-fixation) yielded low nitrogenase activity, and sulfate-reducing bacteria made up 20–50% of the relative diazotrophic abundance in the mangrove forests, indicating that these bacteria might be the major active diazotrophs in this environment.

BIOLOGICAL PROPERTIES OF DIAZOTROPH AZOSPIRILLUM BRASILENSE ISOLATED FROM SPRING TRITICALE ROOTS

Objective. Study the biological properties of the diazotroph Azospirillum brasilense 10/1, promising for improving the nitrogen nutrition of spring triticale and obtaining a high quality crop. Methods. A strain of nitrogen-fixing bacteria A. brasilense 10/1 isolated from washed roots of spring triticale Oberih Kharkivskyi by accumulation cultures method using Dobreiner semi-liquid nitrogen-free medium. Nitrogen-fixing microorganisms were isolated on potato agar with succinic acid by the Dryhalsky method. Potential nitrogenase activity on washed roots of spring triticale plants and nitrogen-fixing activity of azospirilla in pure culture were measured by gas chromatography. Electron microscopic studies of bacterial cells were performed by the method of negative contrast with uranyl acetate. Identification of azospirilla was carried out on the basis of the study of morphological, cultural, physiological and biochemical characteristics and using molecular genetic methods (16S rRNA sequence analysis). The nucleotide sequences were compared with the corresponding sequences from the international database GenBank NCBI using BLAST software. The sensitivity of bacteria to antibiotics and cereal seed pesticides was tested by disk diffusion method. Results. The active strain of nitrogen-fixing bacteria, identified as Azospirillum brasilense 10/1, was obtained by analytical selection methods. The identity of the sequences of 16S rRNA of A. brasilense 10/1 with reference strains of A. brasilense in the GenBank NCBI database is 99.5 % to 99.6 %. Diazotroph A. brasilense 10/1 is sensitive to cefotaxime, norfloxacin, chloramphenicol, gentamicin, erythromycin, kanamycin, furadonin, resistant to polymyxin, ampicillin, oxacillin, amoxicillin, ciprofloxacin, ceftriaxone. Vitavax 200FF and Fundazole dressers do not affect the development of A. brasilense 10/1, Maxim Star 025 FS somewhat inhibits the development of bacteria. Conclusion. The active strain of nitrogen-fixing bacteria A. brasilense 10/1 isolated from washed roots of triticale by methods of analytical selection, is a promising inoculant to increase yields and improve grain quality of this crop. A. brasilense 10/1 is deposited in the Depository of the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine under number B- 7317 and is protected by the patent of Ukraine No. 104212.

Diazotrophic Community Variation Underlies Differences in Nitrogen Fixation Potential in Paddy Soils Across a Climatic Gradient in China.

Biological nitrogen (N2) fixation as a source of new N input into the soil by free-living diazotrophs is important for achieving sustainable rice agriculture. However, the dominant environmental drivers or factors influencing N2 fixation and the functional significance of the diazotroph community structure in paddy soil across a climatic gradient are not yet well understood. Thus, we characterized the diazotroph community and identified the ecological predictors of N2 fixation potential in four different climate zones (mid-temperate, warm-temperate, subtropical, and tropical paddy soils) in eastern China. Comprehensive nifH gene sequencing, functional activity detection, and correlation analysis with environmental factors were estimated. The potential nitrogenase activity (PNA) was highest in warm-temperate regions, where it was 6.2-, 2.9-, and 2.2-fold greater than in the tropical, subtropical, and mid-temperate regions, respectively; nifH gene abundance was significantly higher in warm-temperate and subtropical zones than in the tropical or mid-temperate zones. Diazotroph diversity was significantly higher in the tropical climate zone and significantly lower in the mid-temperate zone. Non-metric multidimensional scaling and canonical correlation analysis indicated that paddy soil diazotroph populations differed significantly among the four climate zones, mainly owing to differences in climate and soil pH. Structural equation models and automatic linear models revealed that climate and nutrients indirectly affected PNA by affecting soil pH and diazotroph community, respectively, while diazotroph community, C/P, and nifH gene abundance directly affected PNA. And C/P ratio, pH, and the diazotroph community structure were the main predictors of PNA in paddy soils. Collectively, the differences in diazotroph community structure have ecological significance, with important implications for the prediction of soil N2-fixing functions under climate change scenarios.

THE INFLUENCE OF BACTERIA OF GENUS AZOSPIRILLUM ON POTENTIAL NITROGENASE ACTIVITY AND BIOSYNTETIC PROCESSES OF SPRING WHEAT AND SPRING TRITICALE

Use of new strain of nitrogen fixing bacteria Azospirillum sp. 77 for inoculation of spring wheat and spring triticale permitted to form an effective associative system diasotroph - plant. The bacterization promoted the reliable increase of potential nitrogenase activity in root zone of plant by 38-220 %, activated the biosynthetic processes, in particular, glutamine synthetase activity increased by 57,0-71,9 %, content of protein in leaves - by 9,7-16,3 %, top of the plants - by 11,9- 18,9 %, weight of the roots - by 7,2-7,3 %.

The effect of tillage systems on some microbial characteristics

Soils were sampled from plots with four variants of tillage methods: (1) conventional tillage (CT); (2) no tillage (NT); (3) minimum tillage (MTS); and (4) no tillage + mulch (NTM). Our aim was to study the influence of tillage on selected soil microbial properties. Determination of urease, dehydrogenase, invertase, arylsulphatase, potential nitrogenase activity, CFU of Azotobacter spp., and carbon microbial biomass has been conducted for time period 2002–2007. Soil samples from 0–0.1 m, 0.1–0.3 m, and 0.3–0.5 m were collected in the spring and autumn. Enzymatic activities (dehydrogenase, urease, arylsulphatase, and invertase) were significantly affected by soil depth and the tillage system employed. The statistically significant higher activity of urease was measured using the minimum tillage system (MTS), compared to the conventional tillage (CT) at soil depths of 0–0.1 m. The highest dehydrogenase activity was measured during the protective tillage treatment (NTM – no tillage with mulch). As far as other enzymatic activities (invertase and arylsulphatase) are concerned, the highest values were recorded in the protective tillage treatments. The highest counts of Azotobacter spp., as well as the highest nitrogenase activity (both statistically significant) were found in the no tillage + mulch (NTM) variant, at depths of 0.1–0.3 m. Microbial biomass (C-biomass) was the highest with the minimum tillage (MTS). The results show a positive influence of protection soil tillage on the reviving of upper layer of topsoil, especially in the variants where soil was supplied with organic matter.

СREATION OF EFFECTIVE ASSOCIATIONS “SPRING WHEAT-DIASOTROPHS OF GENUS AZOSPIRILLUM”

The spring wheat variety Varyag whith high potential nitrogenase activity and the greater contents of chlorophyll in leaves and the active strain of the bacteria Azospirillum sp. 77, selected from the washed roots of the spring wheat variety Varyag, were used for creation of effective association “wheat – diasotrophs of genus Azospirillum”. Inoculation of wheat variety Varyag seeds with the bacterial suspension Azospirillum sp. 77 promoted the reliable increase of nitrogenase activity on roots of plants (by 46 %), content of chlorophyll in leaves (by 18 %) and structural parameters of wheat crop (weight of grain from 1 ear and weight of 1000 grains were increased as a result of inoculation by 16,5 and 16 % accordingly).

Heterotrophic nitrogen fixation in oligotrophic tropical marshes: changes after phosphorus addition

In order to determine the impact of nutrient enrichment on phosphorus (P) limited wetlands, we established experimental P additions in marshes throughout northern Belize. P significantly increased macrophyte primary production, which led to the rapid elimination of cyanobacterial mats. The replacement of cyanobacterial mats by macrophytes constrained autotrophic nitrogen (N) fixation, increased the quantity, and changed the quality of organic matter input to the sediments. We predicted that the activity of sediment heterotrophic N fixers will be impacted by these alterations in carbon input. We used the acetylene reduction technique to measure potential (glucose amended) nitrogenase activity (NA) in sediments from controls and treatment plots that have been P enriched for four years and dominated either by Eleocharis cellulosa, or Typha domingensis for two years. NA in P-enriched plots was 2–3 orders of magnitude higher than NA in controls. NA was positively correlated with the soil reactive P, both total organic and microbial carbon, live root biomass, and total phospholipid fatty acids (PLFA) as an indicator of active microbial biomass. It was negatively correlated with the concentration of ammonium-N. Path analysis revealed that the indirect effect of P on NA through the root biomass was more important than the direct effect of P. NA of the upper sediment layer was consistently higher in Eleocharis than in Typha dominated plots, despite the higher litter input by Typha. We feel that the higher levels of lignin and phenolics occurring in Typha litter, relative to Eleocharis, constrained NA in Typha plots.

The influence of fertilisation and crop rotation on soil microbial characteristics in the long-term field experiment

Soils were sampled from the plots with four variants of fertilisation: Nil – without fertilisation, NPK – mineral fertilisation, FYM – farmyard manure, FYM + NPK – farmyard manure with mineral fertilisation, and two variants of crop rotation: field IV – classical 9-year crop rotation, field B – 2-year rotation of alternative growing. Determination of urease, CFU of <I>Azotobacter</I> spp. and potential nitrogenase activity was conducted during the period 1999–2004. The urease activity was positively affected by manure fertilisation (FYM) and by the combination of FYM + NPK. The statistically significantly highest counts of <I>Azotobacter</I> spp. and the highest nitrogenase activity were determined on field B in variants FYM and FYM + NPK. The results show that there was a higher amount of accessible nitrogen present on field IV than on field B. This might explain the lower counts of <I>Azotobacter</I> spp. and therefore the lower nitrogenase activity. According to our results, activity of urease, CFU of <I>Azotobacter</I> spp. and potential nitrogenase activity are very closely connected with N inputs.

EFFICIENCY OF MICROGUMIN AT COMBINATION AT COMBINATION SEED TREATMENT OF SUMMER BARLEY WITH RAKSIL

The presowing treatment of barley seeds with the microbial preparation of complex action microgumin promotes sufficient increase of this culture yield and intensification of biological activity in rhizoshere. Combined application of microgumin with the raksil fungicide has not affected biological activity of biological preparation. Applied agricultural technology enhances barley seeds’ germination energy, promotes increase of potential nitrogenase activity on plants’ roots and prevents spreading of root rot diseases. It was established that tebuconasol (raksil reactant) in concentrations 0,01; 0,1; 0,3 g/l of nutrient medium promotes increase of nitrogenase activity of the microgumin bioagent Azospіrіllum brasilense 410.

Nitrogen fixation along a north‐south transect in the eastern Atlantic Ocean

Nitrogenase activity, indicative of N2 fixation, was measured in the surface waters along a north‐south transect in the eastern Atlantic Ocean, from Texel (The Netherlands, 53°N) to Cape Town (South Africa, 35°S) using a sensitive on‐line, near real‐time acetylene reduction assay. From the beginning of January to the end of February 2000 nitrogenase activity was detected in varying rates, but only between 14°N and 13°S latitudes. Dark incubations yielded an average activity of 2.2 (± 2.4) µmol m−2 d−1 N, but light increased the activity to 3.7 (± 2.9) µmol m−2 d−1 N. However, nitrogenase activity in the light was sensitive to O2 doubling to 7.6 (± 12.7) µmol m−2 d−1 N when the incubation was anaerobic. In the area where N2 fixation occurred, phosphate concentrations were fourfold lower than in the area where N2 fixation was absent, while silicate levels were higher. The water temperature in the area with N2 fixation was 28°C, while in the adjacent area the temperature was 3°C lower, which might have prevented the proliferation of diazotrophic cyanobacteria. Action spectra revealed that chlorophyll a, phycocyanin, and phycoerythrin are the light‐harvesting pigments supporting nitrogenase activity. In one area in the northern latitudes, potential nitrogenase activity was highest during daytime, which is characteristic for Trichodesmium. In areas with a high potential nitrogenase activity, surface waters were dominated by a phycoerythrin‐containing cyanobacterium. Since nitrogenase activities were highest at night, these cells may have been unicellular cyanobacteria like Crocosphaera.

Nitrogen fixation associated with Suillus tomentosus tuberculate ectomycorrhizae on Pinus contorta var. latifolia.

Tuberculate ectomycorrhizae are a unique form of ectomycorrhiza where densely packed clusters of mycorrhizal root tips are enveloped by a thick hyphal sheath to form a tubercle. The functional significance of such a unique structure has not previously been established. The purpose of the present study was to investigate and measure the potential nitrogenase activity associated with Suillus tomentosus/Pinus contorta tuberculate ectomycorrhizae in two stand ages, young and old, and across a range of nitrogen-poor soil conditions. Short roots were compared with other mycorrhizae and non-mycorrhizal secondary roots using tuberculate ectomycorrhizae. Assessment of nitrogenase activity was determined and quantitative measurements were taken on tuberculate ectomycorrhizae in situ in a variety of different circumstances, by using an adaptation of the acetylene reduction assay. Significant nitrogenase activity was measured associated with S. tomentosus/P. contorta tuberculate ectomycorrhizae whereas no nitrogenase activity was measured with non-tuberculate mycorrhizae or secondary roots without mycorrhizae. Average nitrogenase activity ranged from undetectable to 5696.7 nmol C2H4 g(-1) tubercle 24 h(-1). Maximum nitrogenase activity was 25,098.8 nmol C2H4 g(-1) tubercle 24 h(-1). Nitrogenase activity was significantly higher in young stands than in old stands of P. contorta. Season or some covariate also seemed to affect nitrogenase activity and there was suggestion of a site effect. Suillus tomentosus/P. contorta tuberculate ectomycorrhizae are sites of significant nitrogenase activity. The nitrogenase activity measured could be an important contribution to the nitrogen budget of P. contorta stands. Season and stand age affect levels of nitrogenase activity.

Carbon Dioxide Effects on Heterotrophic Dinitrogen Fixation in a Temperate Pine Forest

Increased net primary productivity (NPP) under elevated atmospheric CO2 requires additional N inputs to sustain C sequestration. We hypothesized that heterotrophic N2 fixation would be stimulated by enhanced litter production under elevated CO2, thus augmenting N availability to plants. To test if N2 fixation is limited by organic substrates alone or in combination with nutrients required for the nitrogenase enzyme, we measured nitrogenase activity (acetylene reduction) in laboratory incubations with water, nutrient, and O2 manipulations. Response of N2 fixation to water, glucose, P, Fe, or Mo was measured under aerobic and anaerobic conditions in forest floor and mineral soil samples from the Duke Forest, NC. Potential nitrogenase activity in forest floor and mineral soil from the Duke Forest Free Air CO2 Enrichment (FACE) site were measured to determine if elevated CO2 enhances N2 fixation. In homogenized slurries with glucose additions, nitrogenase activity was 2 and 400 times greater than controls in organic and mineral soils, respectively (P < 0.01). In laboratory studies, water additions increased N2 fixation 25‐fold in intact soil cores (P < 0.01). Additions of nutrients alone or in combination with C and water did not consistently stimulate N2 fixation in intact soil cores. We detected no CO2 effect on potential nitrogenase activity in Duke FACE soil. Since heterotrophic N2 fixation is not stimulated in temperate pine forests under elevated CO2, additional N assimilation by trees will require increased acquisition of endogenous N, such as increased nutrient use efficiency or enhanced root exploration of the soil.

Does oxygen limit nitrogenase activity in soybean exposed to elevated CO2?

ABSTRACTSoybean (Glycine max L. Merr) plants grown under control (360 µmol mol−1) or elevated CO2 concentration (800 µmol mol−1) from 33 to 42 d after sowing were assayed for various components of in vivo nitrogenase activity to test the hypothesis that increasing carbohydrate supply to nodules would increase the potential (i.e. O2 saturated) nitrogenase activity and impose a more severe O2 limitation on both nodule metabolism and total nitrogenase activity. Within 51 h of elevated CO2 treatment, significant increases relative to control plants were seen in total nitrogenase activity expressed per plant. After 6 d of elevated CO2, the total nitrogenase activity per plant was 18% higher than that in control. This was attributed to an initial increase in nodule size, and a subsequent increase in nodule number following plant exposure to elevated CO2. However, after 9 d of elevated CO2, the potential and total nitrogenase activities per gram nodule dry weight were lower, not higher than corresponding values in plants in the control treatment. These results did not support the hypothesis. It was concluded that the metabolic capacity of the control nodules were not limited by carbohydrate supply, at least at the assay temperatures employed here.

Utilization of the biological nitrogen fixation for soil evaluation

Non-symbiotic nitrogen fixation (potential nitrogenase activity – PNA) of soil samples originating from different plots of long-term field experiments (selected variants: Nil, NPK [mineral fertilisation: 64.6–100 kg N/ha/year], FYM [farmyard manure], and FYM + NPK from three blocks III, IV and B with different crop rotation) was determined in laboratory experiments. The symbiotic nitrogen fixation (total nitrogenase activity – TNA) of the same soil samples was evaluated in hydroponic experiments with pea (2001, 2002) and lucerne (2001) in which the soil samples were used as a natural inoculum. The high values of PNA were found in the variants fertilised with FYM in all three blocks and all experiments. Simultaneously, the variants fertilised with mineral NPK reached low values of PNA. The farmyard manuring enhanced the number of free-living bacteria Azotobacter spp. that were identified in all soil samples. In the hydroponic experiments with pea, the highest nonsignificant values of TNA were found in variants B 284 (FYM + NPK) and III 254 (FYM + NPK) in 2001, and B 214 (FYM) and III 214 (FYM) in 2002. Plants inoculated with soil from these variants formed also high amounts of nodules (significant differences in block IV in 2001) and plant biomass. In the experiments with lucerne, the nonsignificantly highest TNA values were found in variant III 154 (NPK). Variants from block III (214, 254) and IV (114 and 154) showed the nonsignificantly lowest TNA values. The rhizobia that effectuate symbiosis with pea were more active in the soil samples in 2001 than those forming nodules on lucerne.

Diazotrophic bacteria in root-free soil and in the root zone of pine ( Pinus sylvestris L.) and oak ( Quercus robur L.)

Studies on the occurrence, identity and potential nitrogenase activity of diazotrophic bacteria in soil and the root zone of Scots pine ( Pinus sylvestris L.) and common oak ( Quercus robur L.) were carried out. Diazotrophic bacteria were more numerous in soil and the root zone of oak than in those of pine. Most of the bacteria isolated from the roots of both trees belonged to the genera Pseudomonas and Bacillus. The majority of these bacteria had nitrogenase activity (tested by the acetylene reduction assay method) within the range from 4 to 20 nmoles C 2H 4 per culture per hour. No clear relationships between the taxonomic identity and/or source of isolation of the bacteria studied and their nitrogenase activity were found. Biotin, p-aminobenzoic acid (PABA) and yeast extract, applied both separately and in combination, significantly (at least several-fold) stimulated the activity of N 2 fixation in three selected (nitrogenase highly active) bacterial strains, as compared to a control without growth factors.

Role of oxygen limitation and nitrate metabolism in the nitrate inhibition of nitrogen fixation by pea

The impact of nitrate (5–15 mM, 2 to 7 days) on nitrogenase activity and nodule‐oxygen limitation was investigated in nodulated, 21‐day‐old plants of a near‐isogenic nitrate reductase‐deficient pea mutant (A3171) and its wild‐type parent (Pisum sativum L. cv. Juneau). Within 2 days, 10 or 15 mM nitrate, but not 5 mM nitrate, inhibited the apparent nitrogenase activity (measured as in situ hydrogen evolution from nodules of intact plants) of wild‐type plants; none of these nitrate levels inhibited the apparent nitrogenase activity of A3171 plants. Nodule‐oxygen limitation, measured as the ratio of total nitrogenase activity to potential nitrogenase activity, was increased in both wild‐type and A3171 plants by all nitrate treatments. By 3 to 4 days the apparent nitrogenase activity of A3171 and wild‐type plants supplied with 5 mM nitrate declined to 53 to 69% of control plants not receiving nitrate. By 6 to 7 days the apparent nitrogenase activity of A3171 plants was similar to the control value whereas that of the wild‐type plants continued to decline. From 3 to 7 days, no significant differences in nodule‐oxygen limitation were observed between the nitrate (5 mM) and control treatments. The results are interpreted as evidence for separate mechanisms in the initial (O2 limitation) and longer‐term (nitrate metabolism) effects of nitrate on nitrogen fixation by effectively nodulated pea.

Phloem Glutamine and the Regulation of O2 Diffusion in Legume Nodules.

The aim of the present study was to test the hypothesis that the N content or the composition of the phloem sap that supplies nodulated roots may play a role in the feedback regulation of nitrogenase activity by increasing nodule resistance to O2 diffusion. Treating shoots of lupin (Lupinus albus cv Manitoba) or soybean (Glycine max L. Merr. cv Maple Arrow) with 100 [mu]L L-1 NH3 caused a 1.3-fold (lupin) and 2.6-fold (soybean) increase in the total N content of phloem sap without altering its C content. The increase in phloem N was due primarily to a 4.8-fold (lupin) and 10.5-fold (soybean) increase in the concentration of glutamine N. In addition, there was a decline in both the apparent nitrogenase activity and total nitrogenase activity that began within 4 h and reached about 54% of its initial activity within 6 h of the start of the NH3 treatment. However, the potential nitrogenase activity values in the treated plants were not significantly different from those of the control plants. These results provide evidence that changes in the N composition of the phloem sap, particularly the glutamine content, may increase nodule resistance to O2 diffusion and, thereby, down-regulate nodule metabolism and nitrogenase activity by controlling the supply of O2 to the bacteria-infected cells.