Nitrogenase-dependent hydrogen evolution from detached legume nodules and from reaction mixtures containing cell-free nitrogenase has been well established, but the overall effect of hydrogen evolution on the efficiency of nitrogen fixation in vivo has not been critically assessed. This paper describes a survey which revealed that hydrogen evolution is a general phenomenon associated with nitrogen fixation by many nodulated nitrogen-fixing symbionts. An evaluation of the magnitude of energy loss in terms of the efficiency of electron transfer to nitrogen, via nitirogenase, in excised nodules suggested that hydrogen production may severely reduce nitrogen fixation in many legumes where photosynthate supply is a factor limiting fixation. With most symbionts. including soybeans, only 40-60% of the electron fow to nitrogenase was transferred to nitrogen. The remainder was lost through hydrogen evolution. In situ measurements of hydrogen evolution and acetylene reduction by nodulated soybeans confirmed the results obtained with excised nodules. In an atmosphere of air, a major portion of the total electron flux available for the reduction of atmospheric nitrogen by either excised nodules or intact nodulated plants was utilized in the production of hydrogen gas. Some nonleguminous symbionts, such as Alnus rubra, and a few legumes (i.e., Vigns sinensis) apparently have evolved mechanisms of minimizing net hydrogen production, thus increasin their efficiency of electron. transfer to nitrogen. Our res ts indicate that the extent of hydrogen evolution during nitrogen reduction is a major factor affecting the efficiency of nitrogen fixation by many agronomically important legumes. The increasing world population and depletion of fossil fuel supplies have stimulated renewed interest in methods of increasing agricultural productivity while minimizing the consumption of fossil fuels. A major factor limiting agricultural production is nitrogen fertilizer, the synthesis of which consumes major quantities of energy. Approximately 3% (600 X 109 cubic feet; 16.8 X 109 meter3) of the natural gas consumed in the United States in 1973 was used for the synthesis of 17 X 106 U.S. tons (153 X 108 kg) of anhydrous ammonia (1, 2). About 10 X 106 U.S. tons of synthetic ammonia were used for nitrogen fertilizer to supply a portion of an annual agricultural nitrogen demand of about 18 X 106 U.S. tons (1, 2). A large part of the remaining need for nitrogen in agriculture was supplied by nitrogen-fixing organisms, such as legumes, which utilize photosynthetically stored solar energy to reduce atmospheric nitrogen to ammonia. Because the biological nitrogen-fixing process is not dependent upon nonrenewable energy resources, its use in agriculture should be maximized. Factors limiting biological nitrogen fixation therefore deserve thorough investigation (3,4).One characteristic of all cell-free nitrogenase preparations that might limit nitrogen fixation is the release of hydrogen gas concomitant with nitrogen reduction (5-11). During thi...

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 1.11.1.7), dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1), and glutathione reductase (NADPH:oxidized-glutathione oxidoreductase, EC 1.6.4.2). The reactions are essentially the same as those involving scavenging of H 2 O 2 in chloroplasts. Glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) 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.

IntroductionThe maj or source of nitrogen for most higher plants and many microorganisms is nitrate, yet natural mechanisms by which it is reduced have remained obscure. Most of the experimental evidence concerning assimilation of nitrate by higher plants has been included in reviews by NIGHTINGALE (24,25), STREET (29), and VIRTANEN and RAUTANEN (30). The early work of ECKERSON (9) showed that nitrite was formed when the expressed sap of plants was incubated with nitrate and glucose. Since long incubation periods were used in these experiments, the results do not show conclusively whether the nitrate reduction was catalyzed by enzymes from the sap or from contaminating micro6rganisms. Experiments by various investigators (9,12) have demonstrated the formation of nitrite in tissues of nitrogen-deficient plants after nitrate had been added to nutrient solutions and had accumulated in various organs. Nitrite appearance was usually associated with the disappearance of carbohydrates and an increase in respiration. ANDERSON (1) reported the detection of nitrite in leaves and shoots of 25 plant species, and stated that the agent responsible for its formation was thermolabile and oxidizable. B-URSTROM (6), ECKERSON (10), and others have shown that light is involved in nitrate assimilation by the aerial portions of plants. The mechanism of this effect, however, has not been apparent. In a recent study, BHAGVAT (29) reported that the aldehyde oxidase system, which catalyzes the oxidation of aldehyde by oxygen or by nitrate in absence of oxygen, is present in the potato. The physiological importance of this enzyme in nitrate assimilation has not been determined.It is generally concluded (24,25,29,30) that nitrite is a probable intermediate in nitrate metabolism and that the process of nitrate reduction is enzymatic. The characteristics and requirements of a widely distributed enzyme system in higher plants likely to play an important role in nitrate reduction have not been established. It is the purpose of this paper to describe the purification and properties of a pyridine nucleotide-nitrate reductase from soybean leaves already reported from this laboratory (11), and to present evidence for its occurrence in other higher plant species. The crude extract (fraction I) was made by grinding one weight of fresh primary leaves, three weights of cold 0.1 M K2HPO4 buffer (pH 9.0), and two weights of alumina powder (Alcoa A-301) in a Waring Blendor for two minutes at 40 C. The mixture was further ground for three minutes in a Ten Brock homogenizer at 0 to 40 C, then centrifuged in a Servall centrifuge at 20,000 times gravity for 10 minutes at 4°C. The supernatant fluid which was green in color but clear was used for purification. Measurement of activities in homogenates and crude extracts indicated that approximately 10% of the enzyme was dissolved by this procedure. Homogenates used in nutrition and survey experiments were prepared by grinding one weight of fresh tissue with three weights of cold 0.1 M phosphate buffer at pH 7...

Soybean (Glycine max) nodule bacteroids contain high concentrations of poly-,B-hydroxybutyrate and possess a depolymerase system that catalyzes the hydrolysis of the polymer.Changes in poly-p-hydroxybutyrate content Evidence for the metabolic pathway of utilization of BOHB was provided by Sierra and Gibbons (30), who demonstrated that BOHB was oxidized to acetoacetate by crude extracts from M. halodenitrificans. Acetoacetate was further metabolized when ATP, Mg2+, coenzyme A, and oxaloacetate were added to the extracts, suggesting that acetyl-CoA was an intert mediate of the reaction and that utilization occurred through the tricarboxylic acid cycle.The catalysis of nitrogen reduction by cell-free extracts of nitrogen-fixing microogranisms and nodule bacteroids requires a supply of ATP (12,23,25) and an appropriate reductant (4,8, 12,14,25). It has been estimated (3, 10) that 3 to 19 mg of carbohydrate are consumed by legume root nodules for each milligram of N2 fixed. Moreover, two or more carbon atoms are required for the export of each fixed nitrogen atom, in the forms of amino acids and amides, from the nodules to the host (33).It has been established that up to 50% of the dry weight of Rhizobium japonicum bacteroids consists of PHB (14), and that /3-hydroxybutyrate oxidation in in vitro experiments is capable of supplying the electrons for the support of bacteroid nitrogenase activity (14). The purpose of this investigation, therefore, was to study the utilization of PHB in soybean root nodules and to assess the possible role of the polymer as a source of energy for maintenance of nitrogenase activity in nodule bacteroids. MATERIALS AND METHODS CHEMICALSReagent grade chemicals or those of the highest grade available were obtained from commercial sources. NAD, sodium DL-3-hydroxybutyrate, DL-isocitrate lactone (hydrolyzed as recommended by the manufacturer), 2-mercaptoethanol, EDTA, 750 www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

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 cels 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 ree-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.In addition to catalysis of N2 reduction, nitrogenases from all known sources catalyze ATP-dependent H2 evolution. Apparently, H2 evolution during N2 fixation is an inherent property of the nitrogenase reaction. Energy loss through nitrogenase-dependent H2 evolution is important because four or five ATP molecules are consumed per pair of electrons transferred (1, 2) regardless of whether N2 or H+ is the electron acceptor. The significance of ATP-dependent evolution of H2 by nodules as an energy-wasteful process during N2 fixation by legumes has been pointed out by Dixon (3), Schubert and Evans (4) The H2-uptake medium is a further modification of the modified LNB5 medium used to detect acetylene reduction activity in free-living cultures of R. japonicum (11). The H2-uptake medium contains the following in 1 liter of distilled water: NaH2PO4-H20, 150 mg; CaCl2-2H20, 150 mg; MgSO4-7H20, 250 mg; iron EDTA, 28 mg; MnSO4-H20, 10 mg; H3BO3, 3 mg; ZnSO4-7H20, 2 mg; NaMoO4-2H20, 0.25 mg; CuSO4-5H20, 0.04 mg; CoC12-6H20, 0.025 mg; KI, 0