Nitrogenase Activity in Trifolium subterraneum L. in Relation to the Uptake of Nitrate Ions

An experiment was conducted to test the hypothesis that, when nitrogenase and nitrate reductase both contribute to the nitrogen nutrition of a nodulated legume, nitrogenase activity is inversely proportional to the rate of accumulation of organic nitrogen derived from the reduction of nitrate. Trifolium subterraneum L. plants, inoculated with Rhizobium trifolii and sown as small swards, were allowed to establish a closed canopy and steady rates of growth, dinitrogen fixation, and nitrogen accumulation. Swards were then supplied with nutrient solutions of 0, 0.5, 1.0, or 2.5 mm NO₃⁻ with a 29.69% enrichment of ¹⁵N and allowed to grow for a further 33 days. Harvests were made to measure dry weight, nitrogen accumulation, ¹⁵N accumulation, NO₃⁻ content and nitrogenase activity by acetylene reduction assay. Since the ¹⁵N of the plant organic matter could have been derived only from the NO₃⁻ of the nutrient solution, its rate of accumulation provided a measure of the rate of NO₃⁻ reduction. It was found that as this rate increased in response to external NO₃⁻ concentration the rate of nitrogenase activity decreased proportionately. It is concluded that the reduction of nitrate and the reduction of dinitrogen act in a complementary manner to supply a plant with organic nitrogen for growth.     

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR⁻ 108, NR⁻ 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N₂-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR⁻ 108 and NR⁻ 303 contained neither nitrate reductase nor nitrite reductase activities.

Addition of 20 millimolar KNO₃ to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO₃ treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.

The addition of either KNO₃ or KNO₂ to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.

     

The Intracellular Water Activity of Bacteria in Relation to the Water Activity of the Growth Medium

Intracellular water activities ( a _w) calculated from the solute composition of various bacterial cells, are in good agreement with values derived from intracellular freezing point data. Further, and confirming literature results based on freezing points, the intracellular a _w was found to be generally equal to or lower than that of the growth medium.     

Ion Exchange in Sphagnum and its Relation to Bog Ecology

In Sphagnum cuspidatum unesterified polyuronic acids form 12per cent. of the dry weight; in S. acutifolium 25 per cent,of the dry weight. A good correlation has been found for Sphagnabetween the content of unesterified polyuronic acid and thecation exchange ability, and between cation exchange abilityand height of normal habitat above the water table. Anion exchangeability in Sphagna is less than 0.0026 m.eq./g. D.W. comparedwith about 1.2 m.eq./g. D.W. for cations at pH values above7. In natural conditions the exchange sites are, however, onlypartly dissociated. The production of new plant material ina bog dependent on rainwater for nutrients can be sufficientto maintain the pH below 4.5, but on average in England onlyof the order of 2 per cent, of the monovalent cations otherthan H⁺ could be retained in exchangeable form. A greater proportionof polyvalent cations could be retained. The kinetics of cation exchange are consistent with a heterogeneousexchange phase containing regions of high charge density andregions with lower charge density. At equilibrium the proportionsof different cations in the exchange phase are largely explicableby a Donnan distribution, but there are notable exceptions.Two estimates based on Donnan distribution suggest that withlow external pH and/or low cation concentration the apparentconcentration of exchange sites may be 2-3 eq./l., falling withrise in pH and/or increase in cation concentration to 0-9-1-5 eq./l. The apparent dissociation coefficient also variesin these conditions from 2 x 10^–2 to 1 x 10^–4.     

藓类植物传孢类型及其系统演化关系

藓纲包括３个亚纲（Ｂｒｏｔｈｅｒｕｓ,１９２４－１９２５）。泥炭藓亚纲、黑藓亚纲,真藓亚纲,全世界约１００个科,近８００属,约１５０００种。藓类的传孢类型和藓类植物系统演化有密切关系。我们认为传孢不同类型,直接反映了他们的演化程度。我们根据藓类孢子体的分化、蒴齿分化与其机能,分为５种传孢类型。即腐媒传孢型,风媒孢型,汽－风媒传孢型,水媒传孢型,和虫媒传孢型等５种。根据它们的演化程度比较,我们提出了     

Nitrogenase Activity and Amounts of Nitrogenase Proteins in a Frankia-Alnus incana Symbiosis Subjected to Darkness

Effects of prolonged darkness on nitrogenase activity in vivo, nitrogenase activity in vitro, and the amounts of nitrogenase proteins were studied in symbiotic Frankia. Plants of Alnus incana (L.) Moench in symbiosis with a local source of Frankia were grown for 9 to 10 weeks in an 18/6 hour light/darkness cycle. After 12 hours of a light period, the plants were exposed to darkness for up to 40 hours. Nitrogenase activity (acetylene reduction activity) of intact plants was measured repeatedly. Frankia vesicle clusters were prepared from the nodules with an anaerobic homogenization and filtration technique and were used for measurements of in vitro nitrogenase activity and for measurements of the amounts of nitrogenase proteins on Western blots. Antisera made against dinitrogenase reductase (Fe-protein) of Rhodospirillum rubrum and against dinitrogenase (MoFe-protein) of Azotobacter vinelandii were used. Western blots were made transparent and nitrogenase proteins were quantified spectrophotometrically. Nitrogenase activity both in vivo and in vitro decreased after about 23 hours of darkness and continued to decrease to about 25% and 16% of initial activity, respectively, after 40 hours. The amount of Fe-protein and MoFe-protein in Frankia of the same plants decreased to 60% and 35%, respectively, after 40 hours of darkness. Loss of nitrogenase activity thus appeared to be largely explained by loss of MoFe-protein.     

毛萼田菁茎瘤豆血红蛋白含量与固氮活性的关系

在个体瘤发育的不同阶段和植株生长的不同时期,以及在不同的光照条件下,分别测定茎瘤的固氨活性和豆血红蛋白含量。结果表明,毛萼田菁茎瘤的固氨活性和豆血红蛋白含量呈正相关,豆血红蛋白在茎瘤的共生固氮中起着重要的作用。     

Sphagnum Mosses - Masters of Efficient N-Uptake while Avoiding Intoxication

Peat forming Sphagnum mosses are able to prevent the dominance of vascular plants under ombrotrophic conditions by efficiently scavenging atmospherically deposited nitrogen (N). N-uptake kinetics of these mosses are therefore expected to play a key role in differential N availability, plant competition, and carbon sequestration in Sphagnum peatlands. The interacting effects of rain N concentration and exposure time on moss N-uptake rates are, however, poorly understood.We investigated the effects of N-concentration (1, 5, 10, 50, 100, 500 µM), N-form (¹⁵N - ammonium or nitrate) and exposure time (0.5, 2, 72 h) on uptake kinetics for Sphagnum magellanicum from a pristine bog in Patagonia (Argentina) and from a Dutch bog exposed to decades of N-pollution.Uptake rates for ammonium were higher than for nitrate, and N-binding at adsorption sites was negligible. During the first 0.5 h, N-uptake followed saturation kinetics revealing a high affinity (K_m 3.5–6.5 µM). Ammonium was taken up 8 times faster than nitrate, whereas over 72 hours this was only 2 times. Uptake rates decreased drastically with increasing exposure times, which implies that many short-term N-uptake experiments in literature may well have overestimated long-term uptake rates and ecosystem retention. Sphagnum from the polluted site (i.e. long-term N exposure) showed lower uptake rates than mosses from the pristine site, indicating an adaptive response. Sphagnum therefore appears to be highly efficient in using short N pulses (e.g. rainfall in pristine areas). This strategy has important ecological and evolutionary implications: at high N input rates, the risk of N-toxicity seems to be reduced by lower uptake rates of Sphagnum, at the expense of its long-term filter capacity and related competitive advantage over vascular plants. As shown by our conceptual model, interacting effects of N-deposition and climate change (changes in rainfall) will seriously alter the functioning of Sphagnum peatlands.     

Nitrogenase Activity and Photosynthesis in Plectonema boryanum

Nitrogen-starved Plectonema boryanum 594 cultures flushed with N(2)/CO(2) or A/CO(2) (99.7%/0.3%, vol/vol) exhibited nitrogenase activity when assayed either by acetylene reduction or hydrogen evolution. Oxygen evolution activities and phycocyanin pigments decreased sharply before and during the development of nitrogenase activity, but recovered in the N(2)/CO(2) cultures after a period of active nitrogen fixation. Under high illumination, the onset of nitrogenase activity was delayed; however, the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) eliminated this lag. Oxygen was a strong and irreversible inhibitor of nitrogenase activity at low (>0.5%) concentrations. In the dark, low oxygen tensions (0.5%) stimulated nitrogenase activity (up to 60% of that in the light), suggesting a limited but significant respiratory protection of nitrogenase at low oxygen tensions. DCMU was not a strong inhibitor of nitrogenase activity. A decrease in nitrogenase activity after a period of active nitrogen fixation was observed in the N(2)/CO(2-), but not in the A/CO(2-), flushed cultures. We suggest that this decrease in nitrogenase activity is due to exhaustion of stored substrate reserves as well as inhibition by the renewed oxygen evolution of the cultures. Repeated peaks of alternating nitrogenase activity and oxygen evolution were observed in some experiments. Our results indicate a temporal separation of these basically incompatible reactions in P. boryanum.     

Nitrogenase Activity in Response to Darkening and Defoliation of Alnus incana

Huss-Danell, K. and Sellstedt, A. 1985. Nitrogenase activityin response to darkening and defoliation of Alnus incana. —J.exp. Bot. 36: 1352–1358 In the Alnus-Frankia symbiosis the nitrogen-fixing root nodulesare one of the sinks for carbon compounds newly formed in photosynthesisand exported from the leaves (source). The source-sink ratioof cloned plants of Alnus incana was reduced by darkening orby total or partial defoliation and the resulting nitrogenaseactivity (C₂H₂-reduction) was measured. Nitrogenase activityhad nearly ceased 5 h after total defoliation but not untilca. 5 d after total darkening. Most of the activity was lostduring the initial hours and days, respectively. When leaf areawas reduced approximately by half nitrogenase activity decreasedslightly less than by half. Removal of upper leaves seemed lessharmful than removal of lower leaves one day after defoliation.On the following 2 d the treatments appeared to be similar.Thus, nitrogenase activity was largely dependent on newly formedassimilates but could also depend on stored reserves that weremobilized. Measurements of in vitro nitrogenase activity inroot nodule homogenates from darkened plants indicated thatnitrogenase gradually became inactivated and/or depleted after1 and 2 d in darkness Key words: Carbon supply, Frankia, nitrogen fixation     

Diurnal Variations of Nitrogenase Activity in the Field

Nitrogenase activity was determined in situ by repeated assays at regular intervals to compare the patterns of diurnal variations occurring in a rice field, a grassland, and a peanut field. In the rice field only one peak occurred (mid-day), whereas the other systems exhibited typical two-peak patterns, which were suspected to be induced by climatic conditions or possibly by a specific rhythm of exudation. Observed data on diurnal variations in nitrogenase activity were fitted to different models of diurnal variation comprised of computed periodic curves.     

Physiology of Root-Associated Nitrogenase Activity in Oryza sativa

An intact method for measuring immediately linear rates of acetylene reduction was used to investigate the relationship between temperature, pH, O₂ concentration, and light intensity with the rate of root-associated nitrogenase activity in rice (Oryza sativa L.). Nitrogenase activity varied over a temperature range of 10 to 50°C and optimal rates of acetylene reduction were recorded at 35°C. Nitrogenase activity was also influenced by the pH of the liquid surrounding the roots prior to assay. Maximal rates of acetylene reduction were recorded over a pH range from 5.8 to 7.5. Nitrogenase activity was significantly reduced by concentrations of O₂ 0.5% (v/v) or more when the intact plant assay method was used, and no optimum was detected. However, when the plant tops were removed and the cut ends sealed from the atmosphere for 4 hours, acetylene reduction rates were maximal at 0.25% O₂ (v/v). When plants were moved from sunlight (1,400 microeinsteins per square meter per second) to shade (9.6) root-associated nitrogenase activity at 35° C significantly decreased 15 min later to one-fourth the rate and recovered upon return to sunlight. When the light intensity reaching the leaf canopy was progressively reduced from 1,050 to 54 microeinsteins per square meter per second the rate of root-associated nitrogenase activity decreased from 550 ± 135 to 192 ± 55 nanomoles ethylene per gram dry root per hour. The study suggests that the rate of root-associated nitrogenase activity in rice at constant temperature may well be mediated by variations in the concentration of O₂ resulting from changes in the rate of photosynthesis as well as variations in the rate of transport of photosynthate.     

Nitrogenase Activity Associated with Halodule wrightii Roots

Nitrogen fixation (acetylene reduction) associated with roots of the seagrass Halodule wrightii was measured offshore near Beaufort and Moorhead City, N.C. Rates of acetylene reduction were higher in aerobic than in anaerobic assays and were linear for up to 5 days. The temperature range for acetylene reduction was 15 to 35°C with a maximum activity at 35°C. Nitrogenase activity was shown to vary seasonally with highest activities occurring during warmer summer months (23 μg of N₂ fixed per m² per day). At in situ temperature, nitrogenase activities associated with surface-sterilized and non-surface-sterilized roots were similar. One morphological bacterial type was isolated from surface-sterilized roots and identified as Klebsiella pneumoniae type 4B.     

Phosphatase Activity of Anaerobic Organisms

Anaerobic organisms were tested for phosphatase activity in different pH ranges. Several groups of organisms displayed characteristic patterns. Bacteroides fragilis, B. melaninogenicus, and B. ruminicola produced phosphatase with strongest activity at pH 8.6. Fusobacterium mortiferum was the only species of this genus to show strong hydrolysis. The enzyme was active in both acid and alkaline ranges. The activity of gram-positive organisms was variable, the most active groups being Clostridium perfringens, Peptostreptococcus intermedius, P. micros, and Peptococcus constellatus. The incorporation of phosphatase activity into the identification scheme of anaerobes seems feasible. There was a correlation of hydrolysis with several important pathogens.     

The Effect of Temperature on Nitrogenase Activity

Acetylene reduction by detached nodules of four non-legumes(Alnus, Hippopha, Myrica, Casuarina), five legumes (Glycine,Lupinus, Pisum, Vicia, Medicago), and two blue-green algae (Anabaena,Plectonema) was tested with respect to the effect of temperatureon nitrogenase activity. In all cases the activity was sensitiveto temperature change, and with the exception of the legumesthere was a simple exponential response to temperature up tothe optimum. The temperature sensitivity of nitrogenase activityin the two blue-green algae was reduced in low light intensities.Temperature data for several other species are compared, anda simple method of correcting for temperature differences suggested.It is emphasized that allowance for the sensitivity of acetylenereduction to temperature differences must be made if field dataare to be used for purposes of comparison.     

Physiology of Ex Planta Nitrogenase Activity in Rhizobium japonicum

Thirty-nine wild-type strains of Rhizobium japonicum have been studied for their ability to synthesize nitrogenase ex planta in defined liquid media under microaerobic conditions. Twenty-one produced more than trace amounts of acetylene reduction activity, but only a few of these yielded high activity. The oxygen response curves were similar for most of the nitrogenase-positive strains. The strains derepressible for activity had several phenotypic characteristics different from non-derepressible strains. These included slower growth and lower oxygen consumption under microaerobic conditions and lower extracellular polysaccharide production. Extracellular polysaccharide production during growth on gluconate in every nitrogenase-positive strain assayed was lower under both aerobic and microaerobic conditions than the non-derepressible strains. These phenotypic characteristics may be representative of a genotype of a subspecies of R. japonicum. These studies were done in part to enlarge the base number of strains available for studies on the physiology, biochemistry, and genetics of nitrogen fixation.     

自生条件下根瘤菌的氢酶表达与固氮的联系

在自生条件下,研究了根瘤菌的氢酶与固氮酶的共轭表达(coexpression)。氢酶表达受碳源限制和氧耗速率的调节,并为固氮酶表达条件促进,在固氮酶放氢的诱导下而与固氮酶共轭表达。此外,观察到外源氢支持根瘤菌自生固氮的活性,氢支持效应被葡萄糖酸钠和氧抑制。