The methodology, characteristics and application of the sensitive C(2)H(2)-C(2)H(4) assay for N(2) fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N(2)ase-catalyzed reduction of C(2)H(2) to C(2)H(4), gas chromatographic isolation of C(2)H(2) and C(2)H(4), and quantitative measurement with a H(2)-flame analyzer. As little as 1 mumumole C(2)H(4) can be detected, providing a sensitivity 10(3)-fold greater than is possible with (15)N analysis.A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C(2)H(2)-reducing activity in the field. Applications to field samples included an evaluation of N(2) fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N(2) fixation rate of 30 to 33 kg N(2) fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N(2) fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N(2) fixing activity of soybeans. The validity of measuring N(2) fixation in terms of C(2)H(2) reduction was established through extensive comparisons of these activities using defined systems, including purified N(2)ase preparations and pure cultures of N(2)-fixing bacteria.With this assay it now becomes possible and practicable to conduct comprehensive surveys of N(2) fixation, to make detailed comparisons among different N(2)-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N(2) fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N(2) fixation reaction.

The Acetylene-Ethylene Assay for N2 Fixation: Laboratory and Field Evaluation

Biological N2 fixation represents the major source of N input in agricultural soils including those in arid regions. The major N2-fixing systems are the symbiotic systems, which can play a significant role in improving the fertility and productivity of low-N soils. The Rhizobium-legume symbioses have received most attention and have been examined extensively. The behavior of some N2-fixing systems under severe environmental conditions such as salt stress, drought stress, acidity, alkalinity, nutrient deficiency, fertilizers, heavy metals, and pesticides is reviewed. These major stress factors suppress the growth and symbiotic characteristics of most rhizobia; however, several strains, distributed among various species of rhizobia, are tolerant to stress effects. Some strains of rhizobia form effective (N2-fixing) symbioses with their host legumes under salt, heat, and acid stresses, and can sometimes do so under the effect of heavy metals. Reclamation and improvement of the fertility of arid lands by application of organic (manure and sewage sludge) and inorganic (synthetic) fertilizers are expensive and can be a source of pollution. The Rhizobium-legume (herb or tree) symbiosis is suggested to be the ideal solution to the improvement of soil fertility and the rehabilitation of arid lands and is an important direction for future research.

Rhizobium-Legume Symbiosis and Nitrogen Fixation under Severe Conditions and in an Arid Climate

The influence of varied Mg supply (10-1000 micromolar) and light intensity (100-580 microeinsteins per square meter per second) on the concentrations of ascorbate (AsA) and nonprotein SH-compounds and the activities of superoxide dismutase (SOD; EC 1.15.11) and the H2O2 scavenging enzymes, AsA peroxidase (EC 1.11.1.7), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were studied in bean (Phaseolus vulgaris L.) leaves over a 13-day period. The concentrations of AsA and SH-compounds and the activities of SOD and H2O2 scavenging enzymes increased with light intensity, in particular in Mg-deficient leaves. Over the 12-day period of growth for a given light intensity, the concentrations of AsA and SH-compounds and the activities of these enzymes remained more or less constant in Mg-sufficient leaves. In contrast, in Mg-deficient leaves, a progressive increase was recorded, particularly in concentrations of AsA and activities of AsA peroxidase and glutathione reductase, whereas the activities of guaiacol peroxidase and catalase were only slightly enhanced. Partial shading of Mg-deficient leaf blades for 4 days prevented chlorosis, and the activities of the O2.− and H2O2 scavenging enzymes remained at a low level. The results demonstrate the role of both light intensity and Mg nutritional status on the regulation of O2.− and H2O2 scavenging enzymes in chloroplasts.

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Magnesium Deficiency and High Light Intensity Enhance Activities of Superoxide Dismutase, Ascorbate Peroxidase, and Glutathione Reductase in Bean Leaves

Ascorbate peroxidase is a hydrogen peroxide-scavenging enzyme that is specific to plants and algae and is indispensable to protect chloroplasts and other cell constituents from damage by hydrogen peroxide and hydroxyl radicals produced from it. In this review, first, the participation of ascorbate peroxidase in the scavenging of hydrogen peroxide in chloroplasts is briefly described. Subsequently, the phylogenic distribution of ascorbate peroxidase in relation to other hydrogen peroxide-scavenging peroxidases using glutathione, NADH and cytochrome c is summarized. Chloroplastic and cytosolic isozymes of ascorbate peroxidase have been found, and show some differences in enzymatic properties. The basic properties of ascorbate peroxidases, however, are very different from those of the guaiacol peroxidases so far isolated from plant tissues. Amino acid sequence and other molecular properties indicate that ascorbate peroxidase resembles cytochrome c peroxidase from fungi rather than guaiacol peroxidase from plants, and it is proposed that the plant and yeast hydrogen peroxide-scavenging peroxidases have the same ancestor.

Ascorbate peroxidase – a hydrogen peroxide-scavenging enzyme in plants

▪ Abstract The eutrophication of many ecosystems in recent decades has led to an increased interest in the ecology of nitrogen transformation. Chemolitho-autotrophic ammonia-oxidizing bacteria are responsible for the rate-limiting step of nitrification in a wide variety of environments, making them important in the global cycling of nitrogen. These organisms are unique in their ability to use the conversion of ammonia to nitrite as their sole energy source. Because of the importance of this functional group of bacteria, understanding of their ecology and physiology has become a subject of intense research over recent years. The monophyletic nature of these bacteria in terrestrial environments has facilitated molecular biological approaches in studying their ecology, and progress in this field has been rapid. The ammonia-oxidizing bacteria of the β-subclass Proteobacteria have become somewhat of a model system within molecular microbial ecology, and this chapter reviews recent progress in our knowledge of ...

Ammonia-Oxidizing Bacteria: A Model for Molecular Microbial Ecology

The critical problem of oxygen toxicity for nitrogen-fixing organisms may be related to damage caused by oxygen radicals and peroxides An enzymatic mechanism is described for removal of peroxides in root nodules of soybean (Glycine max) The system utilizes ascorbate as an antioxidant and glutathione as a reductant to regenerate ascorbate The enzymes involved are ascorbate peroxidase (ascorbate:hydrogen-peroxide oxidoreductase, EC 11117), dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1851), and glutathione reductase (NADPH:oxidized-glutathione oxidoreductase, EC 1642) The reactions are essentially the same as those involving scavenging of H(2)O(2) in chloroplasts Glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 11119) was not detected During the course of early nodule development, ascorbate peroxidase and dehydroascorbate reductase activities and total glutathione contents of nodule extracts increased strikingly and were positively correlated with acetylene reduction rates and nodule hemoglobin contents The evidence indicates an important role of glutathione, ascorbate, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase as components of a peroxide-scavenging mechanism in soybean root nodules

https://www.pnas.org/content/pnas/83/11/3811.full.pdf

Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules

Some Rhizobium strains synthesize a unidirectional hydrogenase system in legume nodule bacteroids; this system participates in the recycling of hydrogen that otherwise would be lost as a by-product of the nitrogen fixation process. Soybeans inoculated with Rhizobium japonicum strains that synthesized the hydrogenase system fixed significantly more nitrogen and produced greater yields than plants inoculated with strains lacking hydrogen-uptake capacity. Rhizobium strains used as inocula for legumes should have the capability to synthesize the hydrogenase system as one of their desirable characteristics.

https://science.sciencemag.org/content/sci/203/4386/1255.full.pdf

Hydrogenase in Rhizobium japonicum Increases Nitrogen Fixation by Nodulated Soybeans

All aerobic biological systems, including N2-fixing root nodules, are subject to O2 toxicity that results from the formation of reactive intermediates such as H2O2 and free radicals of O2. H2O2 may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN3, CO, and C2H2 were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of ½ Vmax was 70 micromolar for ascorbate and 3 micromolar for H2O2. The high affinity of ascorbate peroxidase for H2O2 indicates that this enzyme, rather than catalase, is responsible for most H2O2 removal outside of peroxisomes in root nodules.

Purification, Properties, and Distribution of Ascorbate Peroxidase in Legume Root Nodules

INTRODUCTION . . ...... ... , . . . . . . ,', . . . . . . . . . , . . . . . . . , . . . . . ,', . . . . . . ,', . . . ,',.,. . . . . . . . . 335 DlHYDROGEN EVOLUTION DURING N2 FIXATION ... . ... . . . .. . . . . ... . . . . .. . . , . . . . . . . , 336 Mechanistic Considerations " . . . . . . . . . . . , . . . . . .. . . . . , . . . . , . . . . . , . . . . , . . . 336 Biological Effects 338 DlHYDROGEN OXIDATION AND CHEMOLITHOTROPHIC GROWTH . . . . . .. . . . . . . . 339 CONSEQUENCES OF H2 OXIDATION ....... ...... ...... . .... ...... . . .. ... . . ... 341 BIOCHEMISTRY OF H2 OXIDATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Com po nent s of the H2 Oxidation System 343 Purification and Properties of Hydrogenase 345 GENETICS OF H2 OXIDATION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 350 Isolation of Uptake Hydrogenase Genes ... . . ...... 351 H up Determinants in Rhizobium leguminosarum ..... . . . .... . . .... . . . .. . . . . . . . . . . . . ... . . . . , 352 Identification of Uptake Hy droge nas e Structural Genes .. .. . ..... . ..... ...... 353 Symbiotic Expression of hup Genes . . ........ . ...... . ...... . . ..... 353 PRACTICAL APPLICATIONS ... , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354

Physiology, Biochemistry, and Genetics of the Uptake Hydrogenase in Rhizobia

A medium is described on which selected Rhizobium japonicum strains express hydrogenase (H2 uptake) activity under free-living conditions. Low concentrations of carbon substrates, decreased oxygen tension, and the quantity of combined nitrogen in the medium were major factors influencing hydrogenase expression. Hydrogenase activity was dependent upon a preincubation period in the presence of H2 under conditions such that the cells did not exhibit nitrogenase activity. H2 uptake rates were easily measured amperometrically in aerobically or anaerobically prepared suspensions from free-living cultures. Six R. japonicum strains that formed nodules with the ability to utilize H2 oxidized this gas when grown in free-living cultures. In comparison six randomly chosen strains forming nodules that lost H2 in air either showed no or low capacity to take up H2 under free-living conditions. The reduction of triphenyltetrazolium chloride in an agar medium was used to detect strains capable of oxidizing H2. This method has enabled us to isolate a spontaneous R. japonicum mutant strain that has lost the ability to utilize H2. This mutant strain forms nodules that evolve H2 but other symbiotic characteristics appear normal. This strain will be useful in evaluating the importance of the hydrogenase system in the nitrogen-fixing process of legumes.

Sterling A. Russell

Papers

Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules

Hydrogenase in Rhizobium japonicum Increases Nitrogen Fixation by Nodulated Soybeans

Purification, Properties, and Distribution of Ascorbate Peroxidase in Legume Root Nodules

Physiology, Biochemistry, and Genetics of the Uptake Hydrogenase in Rhizobia

Expression of hydrogenase activity in free-living Rhizobium japonicum