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1.
Summary 1. Characteristics and methodology of the C 2H 2-C 2H 4 assay for in situ measurement of N 2 fixation are outlined.
2. Electron micrographic analysis of the developmental morphology of the natural soybean symbiosis and C 2H 2-C 2H 4 analysis indicate that increasing N 2-fixing activity from 12–35 days of age is accompanied by an increase in bacteroid number per cell, bacteroid number per vesicle
and inclusions per bacteroid. The mole ratio of leghemoglobin to nitrogenase also increases from 50 to a relatively constant
plateau of 500 to 1500 during this period. The quantitative validity of the C 2H 2-C 2H 4 assay as a measure of N 2 fixation during a complete growth cycle of soybeans on nitrogen-free medium is demonstrated by Σ (C 2H 2→C 2H 4)×28/3 values which are 75–95% of the values determined for N 2 fixed by Kjeldahl analyses.
3. A technique for the establishment of the first callus N 2-fixing symbiosis in mixed cultures of Rhizobium legume provides a defined experimental system for exploration of legume symbiosis. N 2-fixing activity is about 1% of the natural system and is influenced by exogenous auxins and cytokinins. Morphology, including
infection threads and vesicle enclosed bacteroids, is similar to the nodule system.
4. N 2-fixing activity of field-grown soybeans, including varieties which differed in flowering characteristics and maturity dates,
and of peanuts was determined biweekly with the C 2H 2-C 2H 4 assay. Activity extended from nodule initiation to senescence and correlated with the nitrogen demands of the plant and in
most cases >90% of the N 2 fixed during the 60–70 day period of fruit formation and maturation. A logarithmic relationship between N 2-fixing activity and age, and N 2 fixed and age was demonstrated as a fundamental characteristic of these annual symbionts, i.e. log N 2 fixed = k( t−t
0), where t
0 is age at activity initiation. The resultant parameters: 1) age at activity initiation, 2) calculated rate of daily increase
(7–9% for soybeans and 7–10% for peanuts), 3) age at end of logarithmic phase (about 80 days for soybeans), and 4) total N 2 fixed (about 250 mg per soybean plant) are useful bases for evaluation of environmental, bacterial and host effects on N 2 fixation. Various N fertilizers applied at planting and flowering inhibited N 2 fixation of soybeans by decreasing the rate of daily increase.
5. Physical and chemical characteristics of nitrogenase, including those of crystalline Mo-Fe protein, reactions of nitrogenase,
and model studies are consistent with a proposed mechanism.
6. Potential utilities of N 2 fixation research include increased food protein production via initially enhanced N 2 fixation of legumes such as soybeans and eventually extension of N 2-fixing symbioses to non-legumes and new chemistry of N 2, including the direct incorporation of aerial N 2 into important organic compounds.
Contribution No. 1748. 相似文献
2.
An apparatus was designed for simultaneous measurement of rates of N 2 fixation estimated by C 2H 2-C 2H 4 assay (N 2[ C2H2] fixation) and NO 3− absorption by roots of intact, nodulated soybeans ( Glycine max [L.] Merr.). The principal design features include: (a) a gas-tight mist chamber in which nodulated roots can be exposed simultaneously to C 2H 2 in the gas phase and to a liquid phase containing NO 3− sprayed in a fine mist; and (b) provision for sampling the gas phase for C 2H 4 determination, and the liquid phase for NO 3− determination. 相似文献
3.
In field-grown soybeans ( Glycine max L. Merr. cv Harosoy), the percentage of N in the xylem as ureides increased with increasing N 2 fixation. During a 9-week collection period, the ureide content varied from 9.0 to 69.2% of the xylary N. Between 9 and 11 weeks (early pod fill), there was a good correlation ( r = 0.93) between C 2H 2 reduction and the per cent N in xylem as ureides. The per cent N as ureides, however, does not always indicate the reliance of the plant on symbiotic N 2 fixation. This ureide content also depended on the level of NO 3− available to the roots. Non-nodulated soybeans given from 0 to 200 kilogram N per hectare produced xylem sap which averaged from 31.8% to 9.0% N, respectively, in the xylem as ureides over the 9-week period. Feeding of 15N2, 15NH4, or 15NO3 to greenhouse-grown soybeans indicated substantial differences in the initial distribution of N by the xylem stream, but the ultimate distribution of N between plant parts and grain did not vary with available N or percentage of xylary N as ureides. Amino acids, not ureides, were the major source of N in the phloem. The soybeans maintained a similar composition in phloem irrespective of the xylem sap constituents, with N derived from N2, NH4, or NO3 being equally accessible to the phloem stream. 相似文献
4.
Summary Nitrogen fixation by strains of Azospirillum isolated from several rice soils and rice cultivars was investigated by 15N 2 incorporation and C 2H 2 reduction. C 2H 2 reducing ability markedly varied among the strains obtained from soils differing widely in their physico-chemical properties. Large variations in 15N 2 incorporation by Azospirillum isolated from the roots of several rice cultivars were also noticed. The present study reveals that rice cultivars harbour Azospirillum with differential N 2-fixing ability and that plant genotype is of importance for optimal associations. 相似文献
5.
Soil-N (NO 3 ?) initiates as far as a threshold concentration is surpassed manifold physiological reactions on N 2-fixation. Organic N and ammonium oxidised to NO 3 ? means oxygen depletion. Plants suffering under O 2 or infection stress start to excrete ethylene (C 2H 4). C 2H 4 widens the root intercellulars that O 2-respiration will continue. Now microbes may more easily enter the plant interior by transforming the reached methionine into C 2H 4. Surplus nitrate and C 2H 4 inhibit nodulation of leguminous plants. Excess NO 3 ? in the nodulesphere could be diminished by N 2-fixing bacteria which in addition can denitrify or ammonify nitrate. Consequently, it was asked whether C 2H 4 interferes with the potential of N 2-fixing bacteria to reduce nitrate. The groundnut-nodule isolate TNAU 14, from which it was known that it denitrifies and ammonifies nitrate, served as inoculum of a KNO 3-mannitol-medium that was incubated under N 2-, 1% (v/v) N 2?C 2H 4-, and 1% (v/v) N 2?C 2H 2-atmosphere in the laboratory. C 2H 2 was included into the experiments because it is frequently used to quantify N 2-fixing potentials (acetylene reduction array, ARA). Gene-16S rDNA-sequencing and physiological tests revealed a high affiliation of strain TNAU 14 to Rhizobium radiobacter and Rhizobium tumefaciens. Strain TNAU 14 released N 2O into the bottle headspace in all treatments, surprisingly significantly less in presence of C 2H 2. Nitrate-ammonification was even completely blocked by C 2H 2. C 2H 4, in contrast rather stimulated growth, denitrification, and nitrate-ammonification of strain TNAU 14 which consumed the released NH 4 + during continuing incubation. 相似文献
6.
The interaction between the ATP-dependent evolution of H 2 catalyzed by nitrogenase and the oxidation of H 2 via a hydrogenase has been postulated to influence the efficiency of the N 2-fixing process in nodulated legumes. A comparative study using soybean ( Glycine max L. Merr.) cv. Anoka inoculated with either Rhizobium japonicum strain USDA 31 or USDA 110 and cowpea ( Vigna unguiculata L. Walp.) cv. Whippoorwill inoculated with Rhizobium strain 176A27 or 176A28 cultured on a N-free medium was conducted to address this question. Nodules from the Anoka cultivar inoculated with USDA 31 evolved H 2 in air and the H 2 produced accounted for about 30% of the energy transferred to the nitrogenase system during the period of active N 2 fixation. In contrast the same soybean cultivar inoculated with USDA 110 produced nodules with an active hydrogenase and consequently did not evolve H 2 in air. A comparison of Anoka soybeans inoculated with the two different strains of R. japonicum showed that mean rates of C 2H 2 reduction and O 2 consumption and mean mass of nodules taken at four times during vegetative growth were not significantly different. When compared to Anoka inoculated with USDA 31, the same cultivar inoculated with USDA 110 showed increases in total dry matter, per cent nitrogen, and total N2 fixed of 24, 7, and 31%, respectively. Cowpeas in symbiosis with the hydrogenase-producing strain 176A28 in comparison with the same cultivar inoculated with the H2-evolving strain 176A27 produced increases in plant dry weight and total N2 fixed of 11 and 15%, respectively. This apparent increase in the efficiency of N2 fixation for nodulated legumes capable of reutilizing the H2 evolved from nitrogenase is considered and it is concluded that provision of conclusive evidence of the role of the H2-recycling process in N2-fixing efficiency of legumes will require comparison of Rhizobium strains that are genetically identical with the exception of the presence of hydrogenase. 相似文献
7.
Nitrogenase activity (acetylene reduction activity) was found to occur universally in the Cyperus papyrus swamp in Lake Naivasha. Low rates of acetylene reduction activity (0.9–104.9 nmol C 2H 4 g d.wt. roots -1 h -1) were associated with excised roots of C. papyrus but higher rates of activity (89.0–280.4 nmol C 2H 4 g d.wt. roots -1 h -1) were associated with intact root systems of the plant. It was estimated that nitrogen fixation associated with young roots
alone could supply about 26% of the nitrogen requirements of growing papyrus plants. Acetylene reduction activity in the lake
bottom sediments was generally low and associated with adjacent papyrus stands. Plate counts of putative aerobic and facultatively
anaerobic N 2-fixing bacteria associated with papyrus roots showed the presence of high numbers of diazotrophs (5.4 × 10 6 CFU g d.wt. roots -1). Fewer numbers of N 2-fixing bacteria were detected in the sediments (1.9 × 10 3-3.2 × 10 4 CFU g d.wt. sediment -1). 相似文献
8.
Hydrogen production by incubated cyanobacterial epiphytes occurred only in the dark, was stimulated by C 2H 2, and was inhibited by O 2. Addition of NO 3− inhibited dark, anaerobic H 2 production, whereas the addition of NH 4+ inhibited N 2 fixation (C 2H 2 reduction) but not dark H 2 production. Aerobically incubated cyanobacterial aggregates consumed H 2, but light-incubated rates (3.6 μmol of H 2 g −1 h −1) were statistically equivalent to dark uptake rates (4.8 μmol of H 2 g −1 h −1), which were statistically equivalent to dark, anaerobic production rates (2.5 to 10 μmol of H 2 g −1 h −1). Production rates of H 2 were fourfold higher for aggregates in a more advanced stage of decomposition. Enrichment cultures of H 2-producing fermentative bacteria were recovered from freshly harvested, H 2-producing cyanobacterial aggregates. Hydrogen production in these cyanobacterial communities appears to be caused by the resident bacterial flora and not by the cyanobacteria. In situ areal estimates of dark H 2 production by submerged epiphytes (6.8 μmol of H 2 m −2 h −1) were much lower than rates of light-driven N 2 fixation by the epiphytic cyanobacteria (310 μmol of C 2H 4 m −2 h −1). 相似文献
9.
Photoautotrophic growth of a marine non-heterocystous filamentous cyanobacterium, Symploca sp. strain S84, was examined under nitrate-assimilating and N 2-fixing conditions. Under continuous light, photon flux density of 55 μmol photons·m −2 ·s −1 was at a saturating level for growth, and light did not inhibit the growth rate under N 2-fixing conditions even when the photon flux density was doubled (110 μmol photons·m −2 ·s −1). Doubling times of the N 2-fixing cultures under 55 and 110 μmol photons·m −2 ·s −1 were about 30 and 31 h, respectively. Under 110 μmol photons·m −2 ·s −1 during the light phase of an alternating 12:12-h light:dark (L:D) cycle, the doubling time of the N 2-fixing culture was also about 30 h. When grown diazotrophically under a 12:12-h L:D regime, C 2H 2 reduction activity was observed mainly during darkness. In continuous light, relatively large cyclic fluctuations in C 2H 2 reduction were observed during growth. The short-term (<4 h) effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; 5 μM) indicated that C 2H 2 reduction activity was not influenced by photosynthetic O 2 evolution. Long-term (24 h) effects of DCMU indicated that photosynthesis and C 2H 2 reduction activity occur simultaneously. These results indicate that strain S84 grows well under diazotrophic conditions when saturating light is supplied either continuously or under a 12:12-h L:D diel light regime. 相似文献
10.
Rice plants (IR26 and Latisail) obtained at near heading stage from a wetland field were transferred to water culture and exposed to 15N 2 in a gas-tight growth chamber for 7 days to measure N 2-fixing activities associated with the rice. The activities measured varied from 6.5 to 11.6 μmol of N 2 fixed per hill per day. The outer leaf sheath had about 2.5 times higher N 2-fixing activities per unit weight than the root. Slight activities were also found in the basal node and inner leaf sheath. Wrapping basal parts of the stem with aluminum foil did not decrease the activities of N 2 fixation in these parts. Thus, the outer leaf sheath as well as the root are N 2-fixing sites in rice plants. N 2 fixation found in above-ground parts is not due to photoautotrophic organisms. Less than 10% of the fixed nitrogen was translocated from the fixing sites to the leaf blades and the young panicles. 相似文献
11.
Heat evolved by isolated soybean ( Glycine max cv Clark) nodules was measured to estimate more directly the metabolic cost associated with the symbiotic N 2 fixation system. A calorimeter constructed by modifying standard laboratory equipment allowed measurement on 1 gram of detached nodules under a controlled gas stream. Simultaneous gas balance and heat output determinations were made. There was major heat output by nodules for all of the nitrogenase substrates tested (H+, N2, N2O, and C2H2) further establishing the in vivo energy inefficiency of biological N2 fixation. Exposure to a short burst of 100% O2 partially inactivated nitrogenase to permit calculations of heat evolved per mole of substrate reduced. The specific rate of heat evolution for H+ reductions was 171 ± 6 kilocalories per mole H2 evolved in an Ar-O2 atmosphere, that for N2 fixation was 784 ± 26 kilocalories per mole H2 evolved and N2 fixed, and that for C2H2 reduction was 250 ± 12 kilocalories/mole C2H4 formed. When the appropriate thermodynamic parameters are taken into account for the different substrates and products, a ΔH′ of −200 kilocalories per mole 2e− is shown to be associated with active transfer of electrons by the nitrogenase system. These values lead to a calculated N2 fixation cost of 9.5 grams glucose per gram N2 fixed or 3.8 grams C per gram N2, which is in close agreement with earlier calculations based on nodular CO2 production. 相似文献
12.
A mass spectrometer with a membrane-covered inlet was used to measure nitrogen fixation by following changes in the concentration of dissolved N 2 in a stirred suspension of the cyanobacterium Anabaena variabilis in an open system. The results showed a good fit to Michaelis-Menten kinetics with a Km for N 2 of 65 μM at 35°C, corresponding to 0.121 atmosphere of N 2. Corresponding values for the Km for acetylene reduction were 385 μM (0.011 atmosphere at 35°C). Comparison of the values of Vmax for N 2 uptake with those for the acetylene reduction assay under similar conditions gave an average value of 3.8 for the conversion factor between N 2 and C 2H 2 reduction. Reduction of protons to hydrogen was completely inhibited at sufficiently high concentrations of C 2H 2, but even at saturating N 2 concentrations, 1 mol of H 2 was produced for every mole of N 2 reduced. This explains the finding that the observed C 2H 2/N 2 ratio is higher than the value of 3 expected from the requirement for two electrons for acetylene reduction and six for nitrogen reduction. The results correlate well with a mechanism for N 2 reduction involving the equation: N 2 + 8H + + 8e − → 2NH 3 + H 2 which gives a conversion factor between C 2H 2 and N 2 of 4. It is proposed that, in general, 4 is a more appropriate value than 3 for the conversion factor. 相似文献
13.
N 2 fixation by bacteria in associative symbiosis with washed roots of 13 Poaceae and 8 other noncultivated plant species in Finland was demonstrated by the acetylene reduction method. The roots most active in C 2H 2 reduction were those of Agrostis stolonifera, Calamagrostis lanceolata, Elytrigia repens, and Phalaris arundinacea, which produced 538 to 1,510 nmol of C 2H 4·g −1 (dry weight)· h −1 when incubated at pO 2 0.04 with sucrose (pH 6.5), and 70 to 269 nmol of C 2H 4· g −1 (dry weight)·h −1 without an added energy source and unbuffered. Azospirillum lipferum, Enterobacter agglomerans, Klebsiella pneumoniae, and a Pseudomonas sp. were the acetylene-reducing organisms isolated. The results demonstrate the presence of N 2-fixing organisms in associative symbiosis with plant roots found in a northern climatic region in acidic soils ranging down to pH 4.0. 相似文献
14.
Positive effects of legumes and actinorhizal plants on N-poor soils have been observed in many studies but few have been done at high latitudes, which was the location of our study. We measured N 2 fixation and several indices of soil N at a site near the Arctic Circle in northern Sweden. More than 20 years ago lupine ( Lupinus nootkatensis Donn) and gray alder ( Alnus incana L. Moench) were planted on this degraded forest site. We measured total soil N, net N mineralization and nitrification with a buried bag technique, and fluxes of NH +
4 and NO –
3 as collected on ion exchange membranes. We also estimated N 2 fixation activity of the N 2-fixing plants by the natural abundance of 15N of leaves with Betula pendula Roth. as reference species. Foliar nitrogen in the N 2-fixing plants was almost totally derived from N 2 fixation. Plots containing N 2-fixing species generally had significantly higher soil N and N availability than a control plot without N 2-fixing plants. Taken together, all measurements indicated that N 2-fixing plants can be used to effectively improve soil fertility at high latitudes in northern Sweden. 相似文献
15.
Summary The N 2-fixing biota of Macquarie Island are dominated by cyanobacteria growing epiphytically or symbolically with plants or lichens. Highest rates of C 2H 2-reducing activity were found in the leafy lichen Peltigera sp. colonizing herbfields and short grasslands and in the coastal angiosperm Colobanthus muscoides. Significant rates of C 2H 2 reduction were also found to be associated with the liverwort Jamesoniella colorata, commonly occurring in coastal and plateau mires, in a mossbed of Dicranella cardotii colonizing a land-slip face on the grassland slopes at 100 m altitude and within polsters of the mosses Ditrichum strictum and Andreaea sp. found in exposed localities on the plateau at 200–300 m altitude. It was concluded that the common feature of plants supporting active N 2 fixation in dry habitats was the dense packing of stems and leaves, enabling water translocation to the cyanobacterial zone by wick action. Epiphytic cyanobacterial C 2H 2 reduction in wet habitats was widespread and not restricted to any particular plant species. Notable N 2-fixing lichens of the plateau were Pseudocyphellaria delisea and Stereocaulon sp., although both were also occasionally found in coastal herbfields. No significant N 2-fixing activity was associated with any of the dominant grasses tested. Heterotrophic N 2 fixation was also found to be insignificant in the various habitats tested, however N 2-fixing Bacillus ( B. macerans or B. polymyxa) were universally present in coastal, grassland slope, or plateau samples, including moss polster samples. A N 2fixing Clostridium sp. was isolated in only one instance, from soil in the vicinity of a seal wallow on the coast. 相似文献
16.
Summary Aerobic and anaerobic N 2-fixing bacteria developed in the rhizosphere of barley seedlings and exhibited N 2ase activity when seedlings were grown in sterilized sand-nutrient cultures containing low levels of combined nitrogen. The
source of the N 2-fixing bacteria appeared to be the seed. Average daily rates up to 0.9 μmoles C 2H 4 h −1 g −1 dry root tissue were measured, but the intensity of the activity was affected by moisture levels and concentration of combined
N in the rhizosphere. Removal and washing of the roots did not remove the activity, and roots remained active even after surface-sterilization.
An unidentified aerobic N 2-fixing bacterium was isolated from the rhizoplane of active barley roots. Inoculation of barley seedlings with the aerobic
N 2-fixing bacterium enhanced N 2ase activity of excised roots 10-fold, with average rates of 0.9, 1.1 and 1.3 μmoles h −1 g −1 dry root assayed under pO 2 of 0.01, 0.02 and 0.04 atm respectively. The aerobic N 2-fixing bacterium also exhibited N 2ase activity when inoculated into the rhizosphere of oat, rice and wheat seedlings. Microscopic observations of sterilized
live and stained barley roots suggest that the aerobic N 2-fixing bacterium is an endophyte which infects root tissue and metamorphoses into vesicle-like structures. 相似文献
17.
Acetylene reduction, 15N 2 reduction and H 2 evolution were measured in root systems of intact plants of grey alder ( Alnus incana (L.) Moench) in symbiosis with Frankia. The ratios of C 2H 2: 15N 2 were compared with C 2H 2:N 2 ratios calculated from C 2H 2 reduction and H 2 evolution, and with C 2H 2:N 2 ratios calculated from accumulated C 2H 4 production and nitrogen content. It was possible to calculate C 2H 2:N 2 ratios from C 2H 2 reduction and H 2 evolution because this source of Frankia did not show any hydrogenase activity. The ratios obtained using the different methods ranged from 2.72 to 4.42, but these values were not significantly different. It was also shown that enriched 15N could be detected in the shoot after a 1-h incubation of the root-system. It is concluded that the measurement of H 2 evolution in combination with C 2H 2 reduction represents a nondestructive assay for nitrogen fixation in a Frankia symbiosis which shows no detectable hydrogenase activity. 相似文献
18.
A heterotrophic semisolid medium was used with two sensitive assay methods, C 2H 2 reduction and O 2-dependent tritium uptake, to determine nitrogenase and hydrogenase activities, respectively. Organisms known to be positive for both activities showed hydrogenase activity in both the presence and absence of 1% C 2H 2, and thus, it was possible to test a single culture for both activities. Hydrogen uptake activity was detected for the first time in N 2-fixing strains of Pseudomonas stutzeri. The method was then applied to the most-probable-number method of counting N 2-fixing and H 2-oxidizing bacteria in some natural systems. The numbers of H 2-oxidizing diazotrophs were considerably higher in soil surrounding nodules of white beans than they were in the other systems tested. This observation is consistent with reports that the rhizosphere may be an important ecological niche for H 2 transformation. 相似文献
19.
High rates of acetylene (C 2H 2) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C 2H 4 produced per ml per h) and under anaerobic (up to 135 nmol of C 2H 4 produced per ml per h) conditions. Pasteurized effluent developed C 2H 2 reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N 2-fixing Azotobacter and Klebsiella were abundant, and N 2-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability. 相似文献
20.
The effect of photosynthetic photon flux density (PPFD) on nitrogen utilization was determined in peas (Pisum sativum L. cv. Alaska) inoculated with Rhizobium leguminosarum and treated with nutrient solutions containing no combined nitrogen, 16 m M NO 3?, or 16 m M NH 4+. Plants were grown under controlled conditions at three PPFD values ranging from severely limiting to nearly saturating. Carboxylation efficiencies and CO 2-exchange rates were highest in the N 2-fixing plants and lowest in plants supplied with NH 4+, and they generally increased with increasing PPFD. Photoefficiencies increased with PPFD but did not differ appreciably with the form of nitrogen applied. Nitrogen fixation, calculated from C 2H 2-reduction and H 2-evolution data, was inhibited more by NH 4+ than by NO 3?application. Inhibition was counteracted by increasing PPFD. Percentage nitrogen decreased with increasing PPFD in plants treated with combined nitrogen and increased in the plants dependent on N 2 fixation. The data reveal that photosynthetic efficiency and the capacity to fix N 2 in peas are functions of PPFD and the availability of combined nitrogen and that these two factors are interrelated. 相似文献
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