C2H2 Reduction Research Articles

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.

Dinitrogen Fixation Potential and Yield of Hypernodulating Soybean Mutants: A Field Evaluation

Soil N is a major deterrent to increased N2 fixation by soybean [Glycine max (L.) Merr.]. Three hypernodulating mutants (NOD1‐ 3, NOD2‐4, and NOD3‐7) were compared with a nonnodulating mutant (NN5) and the ‘Williams’ parent in a field study in 1988 and 1989 to determine nodulation and agronomic response to 0 and 200 kg N ha−1 applied as urea. Nodule activity was determined by acetylene (C2H2) reduction activity of nodulated roots and relative ureide abundance in vacuum‐extracted xylem sap. In the absence of N fertilizer, two years of data based on the C2H2 reduction assay indicated that all hypernodulating mutants had greater N2 fixation potential than did Williams in early growth stages. Among them, NOD1‐3 had higher N2‐fixing potential (about 10%) than the other two mutants. Both the relative ureide abundance assay (comparing NN5 with other lines in 1989) and the C2H2 reduction assay during the 2‐yr study indicated that N2 fixation by Williams was completely inhibited by N fertilizer through the initial sampling date, while the three hypernodulating mutants showed detectable N2 fixation activity. The relative ureide abundance assay indicated that both NOD1‐ 3 and NOD2‐4 lines were more tolerant of added N fertilizer than was Williams in 1989. In the absence of N fertilizer and at later stages of plant development, N input from N2 fixation by Williams was very similar to that of the three hypernodulating mutants. Seed yields from the hypernodulating mutants were 10 to 30% less than that from Williams in both years. In general, the small differences among soybean lines in seed protein and oil concentrations in both years were not significant. Therefore, total N in harvested seed was less for the hypernodulating lines than for Williams. The results indicate that these hypernodulating mutants are currently inferior to Williams from an agronomic standpoint.

Variation in N 2 Fixation (C 2 H 2 Reduction) Associated with Rhizosheaths of Indian Ricegrass (Stipa hymenoides)

-Relatively low but significant rates of N2 fixation (C2H2 reduction) associated with rhizosheaths of Indian ricegrass Stipa hymenoides varied inversely with percent 02 in test atmospheres, and directly with percent water-holding capacity of sand substrate. Nitrogen fixation was not observed for plants tested in late summer or for nonrhizosheath plants inhabiting nonsandy soil. Delayed linear rates of C2H2 reduction preceded by ca. 2448 h lag periods were observed.

Proteolysis and nitrogen fixation in faba‐bean (Vicia faba) nodules under water stress

Nodulated faba‐beans (Vicia faba L. var. minor) exhibiting high rates of N2 fixation (133 μmol C2H4 g−1 dry weight h−1), were subjected to water restriction. A loss of C2H2 reduction due to water stress was always associated with a decline of the leghemoglobin content for each of the 4 decreasing values of Ψmod. Electron micrographs showed ultrastructural alterations of the fixing tissue, which affected both partners and increased with the severity of water stress. In the nodule cytosol, the alkaline proteolysis approximately doubled when Ψmod decreased from −0.55 MPa to −1.55 MPa. Concomitantly, an increase of the nodule intracellular pH from 6.3 to 7.0 was observed. Proteolysis was due to serine proteases, exhibiting a pH‐optimum of 8 and which actively degraded purified leghemoglobin in vitro (Km=100 μM). The degradation of leghemoglobin during water stress may contribute to the loss of C2H2 reduction and may affect the pattern of recovery upon rewatering.

Nitrogenase Activity by Hapalosiphon flexuosus Associated With Sphagnum erythrocalyx Mats in the Cloud Forest on the Volcano La Soufriere, Guadeloupe, French West Indies

Acid mats of Sphagnum erythrocalyx Hampe. growing on the dome of the tropical volcano La Soufriere, Guadeloupe, French West Indies, support associated populations of the nitrogen-fixing cyanobacterium Hapalosiphon flexuosus Borzi. The acetylene reduction technique was employed to measure nitrogenase activity. The mean rate of C2H4 production in situ by entire plants with the associated cyanobacterium was 19.1 nmol C2H4 gdwh-I (range 0.20101) with a nitrogenase activity for the uppermost green tips 4.5 times greater than that measured for the basal portions. The highest rates for C2H2 reduction were recorded for plants damaged by high winds and for those plants established within vegetation zones recovering from severe damage resulting from the 1976-1977 phreatic eruption of this volcano. The estimated rate of the annual contribution of nitrogen by nitrogen fixation is 4.02 kg N-ha-l year-'. THIS PAPER REPORTS THE RESULTS OF A STUDY OF NIT-ROGEN FIXATION by epiand endophytic cyanobacteria on Sphagnum to the nitrogen economy of the cloud forest orobiome on the tropical volcano La Soufriere. Also discussed is the relationship between the rates of acetylene reduction and the degree of habitat damage following the 1976-1977 eruption of the volcano La Soufriere. La Soufriere (alt. 1467 m), a recently active volcano located on the island of Guadeloupe in the French West Indies (16?40'N, 60?10'E), supports an orobiome (1000-1467 m) termed savane d'altitude (Stehle 1935, 1979), tropical doud forest (Howard 1970), or cloud-shroud ecosystem (sensu Odum 1970). The prevailing doud cover is only occasionally broken by periods of high insolation subjecting the flora, which is adapted to the prevailing low temperatures and insolation values, to unfiltered sunlight and high temperatures (Fritz-Sheridan & Coxson 1988a). Tropical cloud forests, characterized by persistent cloud cover, promote colonization by poikilohydric plants such as mosses and lichens (Grubb 1977, La Bastille & Pool 1978, Weaver et al. 1973, Weaver et al. 1986). The prevailing high moisture and low temperatures reduce the mineralization rate (Grubb 1971, 1977), and mineral loss is accelerated by high rainfall on the steep slopes of La Soufriere, mnaking nitrogen input by fixation a potentially important part of the mineral cycle. I Received 30 January 1990, revision accepted 28 September 1990. 2 Formerly Fritz-Sheridan. The eruptions in 1976 and 1977 disturbed the pre-eruption flora by fumigation with hydrogen sulfide gas and the deposition of ash and boulders, effectively dividing the dome into four distinct posteruption vegetation zones (Howard 1962, Howard et al. 1980, Sastre et al. 1983). This cloud forest supports a diverse group of nitrogen-fixing cyanobacteria, and primary succession in lahar flows on the east flank is by the nitrogen-fixing lichens Dictyonema glabratum and Stereocaulon virgatum (Coxson 1987; Fritz-Sheridan 1987, 1988; Fritz-Sheridan & Coxson 1988a, b; Fritz-Sheridan & Portecop 1987). The occurrence and nitrogenase activities of nitrogen-fixing microepiphytes on dominant species of vascular plants involved in posteruption revegetation (e.g., Clusia mangle, Norantia spiciflora, Tibouchina ornata) have been described by Fritz-Sheridan & Portecop (1987). Sastre et al. (1983) divided the dome of La Soufriere into quadrats and 30 percent of these were occupied by Sphagnum erythrocalyx Hampe.

Nitrogen fixation by Oscillatoria spp. under autotrophic and photoheterotrophic conditions

Summary: Oscillatoria spp. UCSB8 and UCSB25 are both capable of aerobic N2 fixation. The optimum temperature for C2H2 reduction was 22 °C for Oscillatoria sp. UCSB8 and 35 °C for Oscillatoria sp. UCSB25, whilst the optimum temperature for growth on N2 was 25 °C and 30 °C, respectively. In Oscillatoria sp. UCSB25, but not in UCSB8, inhibition of N2 fixation may limit diazotrophic growth at temperatures above 35 °C. When grown under alternating 12 h light and 12 h darkness, both isolates reduced C2H2 predominantly in the dark and both were capable of N2 fixation and photoheterotrophic growth in the presence of 20 μm-DCMU to inhibit photosystem II activity. Under these conditions, the best exogenous carbon source for Oscillatoria sp. UCSB8 was glucose, whilst that for Oscillatoria sp. UCSB25 was fructose. In Oscillatoria sp. UCSB8, exogenous glucose was catabolized mainly through the oxidative pentose phosphate pathway. Although cultures grown photoheterotrophically showed higher specific activities of nitrogenase than photoautotrophic cultures, they grew more slowly. Furthermore, cultures grown photoheterotrophically under alternating light and darkness reduced C2H2 both in the light and in the dark, but the highest rates of C2H2 reduction were observed in the dark. This cyclic pattern of N2 fixation was independent of photosystem II activity.

Effects of Fuel-Reduction Burning on a Eucalyptus obliqua Forest Ecosystem in Victoria

An autumn fuel-reduction burn of low intensity (200-250 kW m-1) was performed in a Eucalyptus obliqua forest near Gembrook, Victoria. The aim of the study was to evaluate the effect of a single burn on floristics, biomass, N content and N2 fixation. The fire burnt 50% of the area in a mosaic pattern, significantly reducing understorey vegetation cover (by 90%) and plant density (by 70%) in burnt areas immediately after the fire. Understorey cover was restored to 40% of the original value 1 year later, but 33% of the understorey species were still absent from burnt areas. In the whole site mosaic, biomass declined by 30 t ha-1 (3 kg m-2) (10%) and N content by 100 kg ha-1 (10 g m-2) (18% excluding soil N, 2% including soil N). These losses were due to significant losses of biomass and N from the understorey only (88%, 85%), standing dead trees (57%, 62%), fallen wood (73%, 60%) and litter (69%, 70%). One year later, there was no significant increase in either biomass or N content. Burnt areas had five times the total nitrogenase activity of unburnt areas, owing to significantly greater specific nitrogenase (C2H2 reduction) activity, three times the nodule weight and 20 times the plant density of unburnt areas for the dominant legume (Pultenaea scabra). Using a calibration ratio for C2H2:N2 of 2.68 :1 derived from glasshouse growth studies, N2 fixation for P. scabra was estimated as 15 g ha-1 year-1 in burnt areas and 3 g ha-1 year-1 in unburnt areas, with a mean of 9 g ha-1 year-' for the whole site mosaic. Adding superphosphate to burnt areas increased estimated N2 fixation significantly by 14%, mainly by increasing nodulation. Losses of N due to the burn (100 kg ha-1) were considerably greater than gains from increases in N2 fixation (6 g ha-1 year-1) one year after the burn. Even allowing for N2 fixation by other, infrequent legumes and greater N2 fixation in subsequent years, these data suggest that the N lost in the burn is more likely to be replaced by inputs from soil reserves and rainwater than from N2 fixation by legumes.

Klebsiella pneumoniae nitrogenase. Mechanism of acetylene reduction and its inhibition by carbon monoxide

The electron flux through the MoFe-protein of nitrogenase from Klebsiella pneumoniae determines the absolute and relative rates of 2H+ reduction to H2 and acetylene (C2H2) reduction to ethylene (C2H4) at saturating levels of reductant (Na2S2O4) and MgATP. High electron flux, induced by a high Fe-protein (Kp2)/MoFe protein (Kp1) ratio, favours C2H2 reduction. These data can be explained if ethylene, the two-electron reduction product of C2H2, is not released until three electrons have been transferred from Kp2 to Kp1. This explanation is also consistent with a pre-steady-state lag phase for C2H4 formation of 250 ms observed when functioning enzyme is quenched with acid. Electron flux through nitrogenase is inhibited by C2H2 at high protein concentrations. This is because the association rate between Kp1 and oxidized Kp2 is enhanced by C2H2, leading to an increased steady-state concentration of the inhibitory complex Kp2oxKp1C2H2. This effect is not relieved by CO. Thus CO and C2H2 (or C2H4) must be bound at the same time to distinct sites, presumably at Mo or Fe centres, on the enzyme.

Nitrogenase in the archaebacterium Methanosarcina barkeri 227

The discovery of nitrogen fixation in the archaebacterium Methanosarcina barkeri 227 raises questions concerning the similarity of archaebacterial nitrogenases to Mo and alternative nitrogenases in eubacteria. A scheme for achieving a 20- to 40-fold partial purification of nitrogenase components from strain 227 was developed by using protamine sulfate precipitation, followed by using a fast protein liquid chromatography apparatus operated inside an anaerobic glove box. As in eubacteria, the nitrogenase activity was resolved into two components. The component 1 analog had a molecular size of approximately 250 kDa, as estimated by gel filtration, and sodium dodecyl sulfate-polyacrylamide gels revealed two predominant bands with molecular sizes near 57 and 62 kDa, consistent with an alpha 2 beta 2 tetramer as in eubacterial component 1 proteins. For the component 2 analog, a molecular size of approximately 120 kDa was estimated by gel filtration, with a subunit molecular size near 31 kDa, indicating that the component 2 protein is a tetramer, in contrast to eubacterial component 2 proteins, which are dimers. Rates of C2H2 reduction by the nearly pure subunits were 1,000 nmol h-1 mg of protein-1, considerably lower than those for conventional Mo nitrogenases but similar to that of the non-Mo non-V nitrogenase from Azotobacter vinelandii. Strain 227 nitrogenase reduced N2 at a higher rate per electron than it reduced C2H2, also resembling the non-Mo non-V nitrogenase of A. vinelandii. Ethane was not produced from C2H2. NH4+ concentrations as low as 10 microM caused a transient inhibition of C2H2 reduction by strain 227 cells. Antiserum against component 2 Rhodospirillum rubrum nitrogenase was found to cross-react with component 2 from strain 227, and Western immunoblots using this antiserum showed no evidence for covalent modification of component 2. Also, extracts of strain 227 cells prepared before and after switch-off had virtually the same level of nitrogenase activity. In conclusion, the nitrogenase from strain 227 is similar in overall structure to the eubacterial nitrogenases and shows greatest similarity to alternative nitrogenases.

Comparison between isotope dilution and acetylene reduction methods to estimate N2 fixation rate of white clover in grass/clover swards

AbstractIn two field experiments acetylene (C2H2) reduction by white clover in mixed swards was compared to N2‐fixed measured by 15N dilution. In both experiments, samples for C2H2 reduction were 7.5 cm diameter turves taken from plots within which microplots of 24 cm diameter were delimited and to which 15N was applied as ammonium sulphate (15NH4)2SO4). C2H2 reduction was assayed every 6–7 d. The rate of C2H2 reduction per unit length of stolon was applied to the estimated stolon length within the appropriate microplot at the time of assay, and the amount of C2H2 that would have been reduced within the microplot was estimated by integration.In experiment 1, turves taken from grass/clover swards to which 0, 1·5, 3·0, 4·5 or 6·0 g N m−2 had been applied were incubated in sealed chambers (10% C2H2, 90% air). The mean ratio of C2H2 reduced to N2 fixed during 5 weeks was 0.74:1. Application of N fertilizer lowered the proportion of assimilated N derived from N2 fixation from 95% in unfertilized swards to 83% in those receiving 6 g N m−2 (60 kg N ha−1). In experiment 2, clover roots and stolons from plots that previously had been grazed were dissected from turves and incubated in a stream of C2H2 and air (i.e. the open system). The maximum rate of ethylene (C2H4) produced during the first 12 min was taken as a measure of true nitrogenase activity. The relationship between C2H2 reduced and N2 fixed was significant (r=0.80**). The mole ratio was 0.55:1 for the 6 weeks duration of the experiment, the low ratio possibly being due to disturbance of the nodules adversely affecting acetylene reduction. Mole ratios from both experiments were well short of the theoretical 4·3:1. Using the open system does not, therefore, overcome the shortcomings of the acetylene reduction technique for measuring N2 fixation of white clover in mixed swards.

Nitrogen Fixation of Nodulation Mutants of Soybean as Affected by Nitrate

It was previously reported that three soybean (Glycine max [L.] Merr.) nodulation mutants (NOD1-3, NOD2-4, and NOD3-7) were partially tolerant to nitrate when nitrate was supplied simultaneously with inoculation at the time of transplanting. The current study evaluated the effect of short-term nitrate treatment on nitrogenase activity (C(2)H(2) reduction per plant and per nodule weight) and on relative abundance of ureides when nitrate application was delayed until plants were 3 weeks old and nodules were fully developed. Nitrogenase activity of the mutants was similar to that of Williams after an initial 3-week growth period, prior to nitrate treatment. Application of 5 millimolar nitrate resulted in greater inhibition of nitrogenase activity in Williams than in the three mutants. NOD1-3 was most tolerant of nitrate among the mutants tested and showed the highest relative abundance of ureides. Although C(2)H(2) reduction activity per plant for NOD1-3 was higher than for Williams in the presence of nitrate, C(2)H(2) reduction activity per gram of nodules was lower for NOD1-3 than for Williams in the presence and absence of nitrate. Compared to Williams, NOD1-3 had higher nodule ureide concentration and had similar glutamine synthetase activity in nodule tissue, indicating its nodules have normal nitrogen assimilation pathways. Nitrate application resulted in ureide accumulation in nodule tissue as well as in all plant parts assayed. Unexpectedly, nitrate treatment also increased the rate of ureide degradative capacity of leaves in both NOD1-3 and Williams. The data confirmed that nitrogenase activity of the selected nodulation mutants was more, but still only partially, tolerant of nitrate compared with the Williams parent.

Analysis of Azotobacter vinelandii nitrogenase reaction by monoclonal antibodies.

Nitrogenase catalyzes not only the reduction of N2 to NH3 but also the reduction of C2H2 to C2H4 and H+ ion to H2 gas, etc. The detailed mechanism of the nitrogenase reaction is not clear. We have prepared monoclonal antibodies against Component I nitrogenase of A. vinelandii and examined the effects of antibodies on the nitrogenase reactions. A monoclonal antibody designated MA-1 inhibited C2H2 reduction activity strongly but did not inhibit H2 evolution activity. MA-2, on the contrary, inhibited only H2 evolution activity. MA-8 inhibited both C2H2 reduction and H2 evolution activity to the same extent.

Effects of temperature regime and fertilization on nitrogenase activity of black alder seedlings during autumn in Illinois, U.S.A.

A pot experiment was conducted to determine the effects of two regimes of temperature and fertilization on nitrogenase activity (acetylene reduction) of naturalized black alder (Alnusglutinosa (L.) Gaertn.) seedlings during autumn in central Illinois, United States. An equal number of Frankia-nodulated plants were fertilized with either a complete or a nitrogen-free fertilizer solution. After 3 months of growth in a glasshouse, half of the plants in each fertilization group were transferred outside in mid-September. Rates of C2H2 reduction decreased throughout autumn for plants in all treatments, although exposure to freezing temperatures accelerated the decline of nitrogenase activity. Alders that did not receive nitrogen fertilization were still reducing C2H2 under natural light conditions in the heated glasshouse on December 10 at a rate of 7.5 nmol•mg−1•h−1. Compared with alders not receiving mineral nitrogen, complete fertilization resulted in a 45% reduction of initial autumnal rates of C2H2 reduction and early inactivity of nitrogenase. Leaf senescence and leaf abscission of naturalized black alder were not induced by decreasing photoperiod alone in Illinois. We conclude that N2 fixation of black alder in Illinois can proceed late into the autumn as long as green leaves remain attached to the plant and nodules are not subjected to low and freezing temperatures.

Glycolate metabolism in cyanobacteria. III. Nitrogen controls excretion and metabolism of glycolate in Anabaena cylindrica

The effect of nitrogen on excretion and metabolism of glycolate in Anabaena cylindrica (CCAP 1403/2a) was studied. Glycidate, an inhibitor of glutamate:glyoxylate aminotransferase (EC 2.6.1.4), reduced the L‐methionine‐DL‐sulfoximine‐induced NH4+ release by ca 40%, while net CO2 fixation and C2H2 reduction were not lowered. This indicates that at least a part of the glyoxylate synthesized in A. cylindrica is metabolized via glycine to serine. Addition of NH4Cl or glutamate to the medium reduced the excretion of glycolate. At pH 9, under air, NH4Cl reduced the excretion by 10–30% and under high pO2 (0.03 kPa CO2 in O2) by about 80–90%. At pH 7.5, under high pO2, NH4Cl and glulamate reduced the excretion by about 40 and 80%, respectively. Also, the presence of NH4Cl stimulated the animation of glyoxylate under such conditions as shown by an increased glycine pool and a decreased glutamate pool. We suggest that nitrogen regulates the capacity of A. cylindrica to retain and recycle glycolate intracellularly and that glutamate serves as an amino donor in the conversion of glyoxylate to glycine.

Asymbiotic nitrogen fixation and denitrification in a mature forest in coastal British Columbia

Gaseous nitrogen inputs due to asymbiotic nitrogen fixation and outputs due to biological denitrification were estimated for a mature mid-successional Pseudotsugamenziesii (Mirb.) Franco–Thujaplicata Donn–Tsugaheterophylla (Raf.) Sarg. forest in southwestern British Columbia. Forest floor material, mineral soil, decaying wood, foliage, and bark were incubated in an atmosphere of 10 kPa C2H2 to allow the simultaneous measurement of N2O production by denitrifying bacteria and C2H2 reduction by free-living bacteria and blue-green algae. Forest floor material accounted for 70% of an estimated total annual nitrogen fixation of 0.3 kg N•ha−1•year −1.•Relatively small amounts of nitrogen were fixed in mineral soil, decaying wood, and foliage, and no indication of nitrogen fixation activity in bark was detected. Some denitrification was found; it was essentially negligible, although possibly underestimated. The net gaseous nitrogen input into the mid-successional forests of the study area is likely to be < 5% of the total net nitrogen inputs, which are primarily from precipitation.

The Glycine‐Glomus‐Bradyrhizobium symbiosis. IX. Nutritional, morphological and physiological responses of nodulated soybean to geographic isolates of the mycorrhizal fungus Glomus mosseae.

The objective of the work was to determine differences in plant response to geographic isolates of a vesicular‐arbuscular mycorrhizal (VAM) fungus, and to demonstrate the need for such determinations in the selection of desirable host‐endophyte combinations for practical applications. Soybean (Glycine max (L.) Merr.) plants were inoculated with Bradyrhizobium japonicum and isolates of the VAM‐fungal morphospecies Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe, collected from an arid (AR), semiarid (SA) or mesic (ME) area. Inoculum potentials of the VAM‐fungal isolates were determined and the inocula equalized, achieving the same level of root colonization (41%, P>0.05) at harvest (50 days). Plants of the three VAM treatments (AR, SA and ME) were evaluated against von VAM controls. Significant differences in plant response to colonization were found in dry mass, leaf K, N and P concentrations, and in root/shoot, nodule/root, root length/leaf area and root length/root mass ratios. The differences were most pronounced and consistent between the AR and all other treatments. Photosynthesis and nodule activity were higher (P<0.05) in all VAM treatments, but only the AR plants had higher (P<0.05) photosynthetic water‐use efficiency than the controls. Nodule activity, evaluated by H2 evolution and C2H2 reduction, differed significantly between treatments. The results are discussed in terms of nutritional and non‐nutritional effects of VAM colonization on the development and physiology of the tripartite soybean association in the light of intraspecific variability within the fungal endophyte.

Effect of Oxygen and Malate on NO3− Inhibition of Nitrogenase in Soybean Nodules

Soybean (Glycine max cv Hodgson) nitrogenase activity (C(2)H(2) reduction) in the presence or absence of nitrate was studied at various external O(2) tensions. Nitrogenase activity increased with oxygen partial pressure up to 30 kilopascals, which appeared to be the optimum. A parallel increase in ATP/ADP ratios indicated a limitation of respiration rate by low O(2) tensions in the nodule, and the values found for adenine nucleotide ratios suggested that the nitrogenase activity was limited by the rate of ATP regeneration. In the presence of nitrate, the nitrogenase activity was low and less stimulated by increased pO(2), although the nitrite content per gram of nodules decreased from 0.05 to 0.02 micromole when pO(2) increased from 10 to 30 kilopascals. Therefore, the accumulation of nitrite inside the nodule was probably not the major cause of the inhibition. Instead, inhibition by nitrate could be due to competition for reducing power between nitrate reduction and bacteroid or mitochondrial respiration inside the nodule. This is supported by the observation of decrease in ATP/ADP ratios from 1.65, in absence of nitrate, to 0.93 in the presence of this anion at 30 kilopascals O(2). Furthermore, the inhibition was suppressed by the addition, to the plant nutrient solution, of 15 millimolar l-malate, a carbon substrate that is considered to be the major source of reductant for the bacteroids in the symbiosis.

Occurrence ofAzospirillum spp. in coconut-based farming systems

Occurrence of Azospirillum was investigated in coconut-based farming systems, such as high-density multispecies cropping (15 crops), multi-storeyed cropping (3 crops), mixed cropping with tea and coffee (2 crops), intercropping with tropical tubers (5 crops), mixed farming with grasses (3 crops) and in 3 crops, arecanut,Mimosa invisa and sugarcane from other plots. A total of 26 plantation crops and intercrops were included in the study. Incidence of Azospirillum was determined by 2,3,5-triphenyl-tetrazolium chloride reduction and by culturing root fragments in N-free semisolid malate medium. Root samples from guava, mango and mimosa did not show any tetrazolium reduction or sub-surface pellicular growth. The extent of occurrence of Azospirillum seemed to depend upon the crop combinations. In a mixed farming system where guinea grass was one of the component crops, more root fragments of coconut and pepper demonstrated tetrazolium reduction activity than when guinea grass was absent.Azospirillum lipoferum andA. brasilense constituted 42% and 45% of the isolates, respectively, in the coconut-based cropping systems. Isolates from guinea grass, sugarcane and jackfruit exhibited higher nitrogenase (C2H2 reduction) than those isolated from plantation crops, tuber crops and spices. The large variation in the extent of association and nitrogenase activity of isolates from different crops indicated the need for inoculation with efficient cultures in a number of crops in coconut-based cropping systems.

Nitrite, a new substrate for nitrogenase.

We have examined the reactivity of the purified component proteins of Azotobacter vinelandii nitrogenase (Av1 and Av2) toward nitrate and nitrite. Nitrate has no effect on H2 evolution or C2H2 reduction by nitrogenase and thus is neither a substrate nor an inhibitor. Nitrite dramatically inhibits H2 evolution. This inhibition has two components, one irreversible and one reversible upon addition of CO. The irreversible inhibition is due to nitrite inactivation of the Fe protein. The rate of this inactivation is greatly enhanced by addition of MgATP, suggesting the [4Fe-4S] cluster is the site of nitrite attack. The reversible inhibition does not represent an inhibition of electron flow but rather a diversion of electrons away from H2 evolution and into the six-electron reduction of nitrite to ammonia. Thus, nitrogenase functions as a nitrite reductase.

Supplemental nitrogen fertilizer to soybeans. III. Effects on nitrogen fixation.

開花期窒素追肥がダイズの窒素固定能や窒素同化に及ぼす効果を調査した. さらに, 窒素追肥を行なったダイズを用い, アセチレン還元能の経時的測定を通じてダイズの窒素固定量を推定する可能性についても検討した. 重窒素(~<15>N)でラベルした窒素肥料を用い, 砂耕栽培でダイズを肥料窒素と固定窒素のみで生育させた. アセチレン還元能の経時的積分値と作物体の15N希釈率から算定した窒素固定量との比を求めた. その値, すなわち前者から後者への換算係数は1.05であった. ポットで土耕栽培したダイズでは, 開花期窒素追肥をする事によって, 根粒重と単位根粒重当りの窒素固定比活性とも低下した. しかし, 比活性は比較的早く回復し, 窒素追肥による窒素固定阻害には, 根粒重の減少の影響の方が大きかった. 前述の換算係数を用い, 開花期窒素追肥を行なったダイズの窒素固定量を推定した. 窒素追肥による窒素固定減少量はかなり大きかった. しかし, 追肥窒素吸収量は, 追肥による窒素固定減少量よりも多く, また, 両者の差は追肥量に応じて増加した. 3.82gN/個体を追肥した場合, その吸収量が1.60gN/個体であるのに対し窒素固定減少量は1.01gN/個体にとどまった. 窒素追肥量の増加に伴う窒素固定量と追肥窒素吸収量の和の増加は, 全窒素同化量の増加にほぼ見合うものであり, アセチレン還元法に基づく本定量法の有効性が窺えた.