The Influence of Light and Oxygen on Nitrogenase Activity in the Lichen Stereocaulon paschale

Thalli of the lichen Slereocaulon paschale (L.) Fr. were prctreated in the light (light activated) or in the dark (dark starved). In short-time experiments with both light activated arid dark starved thalli, the nitrogenasc activity was higher in the light than in the dark, Light activated thalli had a very much higher rale of C₂H₂ reduction than dark starved thalli, both in the light and in the dark. The dark starved lhalli showed increasing nilrogenase activity when incubated in the light. Either light or oxygen was necessary for nitrogenase activity in light activated thalli. and up to about 10kPa oxygen they showed additive effects. Both in the light and in the dark the nitrogenase activity decreased when the oxygen partial pressure was lower than in normal air. The experimental data thus showed a short-term effect of light on nilrogenase activity by provision of ATP and reductant, and a long term effect probably by build up of reserves that were later utilized. Any immediate effect of photorespiration on nitrogenase activity could not be found in light activated thalli.     

Growth and nitrogen relations in the mat-forming lichens Stereocaulon paschale and Cladonia stellaris

BACKGROUND AND AIMS: Mat-forming lichens in the genera Stereocaulon and Cladonia have ecosystem-level effects in northern boreal forests. Yet the factors affecting the productivity of mat-forming lichens are not known. The aim of the presented work was to investigate whether mat-forming lichens adapted to low N availability employ N-conserving mechanisms similar to those of vascular plants in nutrient-poor ecosystems. Specifically, the following questions were asked: (a) Do lichens translocate N from basal areas to apical growth areas? (b) Are the quantities of N translocated of ecological significance. (c) Is lichen growth dependent on tissue N concentration [N]. METHODS: Two different, but complementary, field experiments were conducted using the mat-forming N2-fixing Stereocaulon paschale and non-fixing Cladonia stellaris as model species. First, N translocation was investigated by feeding lichens with Na(15)NO3 either directly to the apex (theoretical sink) or to the basal part (theoretical source) and observing the redistribution of (15)N after a growth period. Secondly, growth and variation in [N] in thalli of different lengths was measured after a growth period. KEY RESULTS: (15)N fed to lower parts of lichen was translocated towards the growing top, but not vice versa, indicating physiologically dependent translocation that follows a sink-source relationship. In the growth experiment where thalli were cut to different lengths, the significant decrease in [N] in apices of short vs. longer thalli after a growth period is consistent with internal relocation as an ecologically important source of N. CONCLUSIONS: The presented results demonstrate that internal recycling of N occurs in both species investigated and may be ecologically important in these mat-forming lichens under field conditions. The higher nitrogen use efficiency and relative growth rate in C. stellaris in comparison with S. paschale probably enable C. stellaris to dominate the ground cover vegetation in dry boreal coniferous forests under undisturbed conditions.     

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.     

Water Storage in the Lichen Family Umbilicariaceae

Quantitative relationships between thallus structure and water storage and retention capacities In 12 species of the lichen family Umbilicariaceae were explored using three recent techniques for plant structure analysis: stereology (3D quantification of microscopic Images), mercury Intrusion porosimetry (determination of pore size distribution of tissues) and low-temperature scanning electron microscopy (LTSEM). Water storage capacity of the thallus was related neither to thallus density nor surface area of the thallus; It was directly related to the total porosity of the thallus and inversely related to the proportion of thallus volume occupied by cell walls and gelatinous substances. Water retention capacity increased with increasing thallus density and was decreased by slight increases in the surface area of the upper side of the thallus. Water storage and retention capacities exhibited a positive correlation only when the storage capacity was expressed on a surface area basis. LTSEM study of fully hydrated specimens revealed that many intercellular spaces of the upper cortex and upper parts of the algal layer contained liquid water. Intercellular spaces of the lower part of the algal layer and medulla were in general either airfilled or partially occupied by gelatinous substances. Species with rhizinomorphs and substrate-hygrophytic (water uptake from surface run-offs) stored more water and retained it longer than aerohygrophytic species (water uptake from the atmosphere) lacking rhizinomorphs. Thallus structure of aerohygrophytic species seems to facilitate rapid gas exchange with the environment, improving water uptake and carbon gain when atmospheric moisture is available but accelerating dehydration when the atmosphere becomes dry.     

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.     

Biology of Azospirillum-Sugarcane Association: Enhancement of Nitrogenase Activity

Azospirillum brasilense was reisolated from associations with callus tissue cultures of sugarcane and compared with stock cultures of the inoculated bacterium and related strains. Although the reisolate had a growth rate similar to stock cultures, it exhibited a severalfold increase in maximum specific activity of nitrogenase. The reisolate and the parent culture had similar ultrastructure. The general ultrastructure of Azospirillum is described. The bacterium was capsulated when grown on nitrogen-free nutrient agar plates and on callus, but was not capsulated when growing in a subsurface zone in N-free semisolid nutrient agar, except rarely in aging cultures. Capsulation may be a protective mechanism against unfavorable pO₂ under dinitrogen-fixing conditions. Pleomorphism occurred in capsulated forms, and the ultrastructure of these forms is described.     

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.     

南岭黄檀根瘤固氮酶和吸氢酶活性研究

测定不同环境条件下南岭黄檀根瘤固氮酶和吸氢酶活性。结果表明,南岭黄檀根瘤具有吸氢酶活性,外源Ｈ＿２可提高固氮酶活性（２５％）,表明吸氢酶有助于提高固氮效率。低浓度的硝酸盐（２０ｍｇ。１￣＿（－１）不影响根瘤固氮酶和吸氢酶活性,但浓度达４０ｍｇ·ｌ￣（－１）时有抑制作用,而铵盐在上述浓度下均表现抑制作用。离体根瘤固氮酶和吸氢酶活性表达的最适温度为２５℃,过高或过低均有抑制作用。土壤含水量及光照强度均明显影响其固氮酶和吸氢酶活性。     

Diazotrophy and Nitrogenase Activity in the Archaebacterium Methanosarcina barkeri 227

Nitrogen fixation (diazotrophy) has recently been demonstrated in several methanogenic archaebacteria. To compare the process in an archaebacterium with that in eubacteria, we examined the properties of diazotrophic growth and nitrogenase activity in Methanosarcina barkeri 227. Growth yields with methanol or acetate as a growth substrate were significantly lower in N₂-grown cultures than in NH₄⁺-grown cultures, and the culture doubling times were increased, indicating that diazotrophy was energetically costly, as it is in eubacteria. Growth of nitrogen-fixing cells was inhibited when molybdenum was omitted from the medium; addition of 10 nM molybdate stimulated growth, while 1 μM molybdate restored maximum diazotrophic growth. Omission of molybdenum did not inhibit growth of ammonia-grown cells. Tungstate (100 μM) strongly inhibited growth of molybdenum-deficient diazotrophic cells, while ammonia-grown cells were unaffected. The addition of 100 nM vanadate or chromate did not stimulate diazotrophic growth of molybdenum-starved cells. These results are consistent with the presence of a molybdenum-containing nitrogenase in M. barkeri. Acetylene, the usual substrate for assaying nitrogenase activity, inhibited methanogenesis by M. barkeri and consequently needed to be used at a low partial pressure (0.3% of the headspace) when acetylene reduction by whole cells was assayed. Whole cells reduced 0.3% acetylene to ethylene at a very low rate (1 to 2 nmol h⁻¹ mg of protein⁻¹), and they “switched off” acetylene reduction in response to added ammonia or glutamine. Crude extracts from diazotrophic cells reduced 10% acetylene at a rate of 4 to 5 nmol of C₂H₄ formed h⁻¹ mg of protein⁻¹ when supplied with ATP and reducing power, while extracts of Klebsiella pneumoniae prepared by the same procedures had rates 100-fold higher. Acetylene reduction by extracts required ATP and was completely inhibited by 1 mM ADP in the presence of 5 mM ATP. The low rates of C₂H₂ reduction could be due to improper assay conditions, to switched-off enzyme, or to the nitrogenase's having lower activity towards acetylene than towards dinitrogen.     

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.     

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。     

Nitrogenase Activity Measurements in Intact Plants of Alnus incana

A technique for C₂H₂-reduction assay on intact plants of Alnus incana (L.) Moench was evaluated. Cloned plants were grown, in pots, on fine gravel. During assay only the pot was inserted into a Perspex incubation chamber of simple construction. The incubation volume was rather small, plants with various shoot heights could be used, and the shoot was not exposed to the C₂H₄ produced. Intact plants showed high and constant C₂H₂-reduction rates during several hours of incubation. In comparison, excised nodulated roots conventionally incubated in test tubes showed low and decreasing rates, due to removal of the photo-synthesizing shoot and injury to the root nodules when drawn from the pot. Repeated nitrogenase activity assays on the same intact individual plants did not affect growth. The technique thus proved useful in studies. where repeated nitrogenase activity measurements are important.     

Potassium and the Recovery of Arterial Smooth Muscle after Cold Storage

The influence of K on the performance of vascular smooth muscle was studied by observing the mechanical performance of the muscle under conditions in which the magnitudes of [K_i] and of the [K_i]:[K_o] ratio varied in opposite directions. During prolonged storage at 4°C the artery strips lost K and their ability to respond to stimuli. Subsequently they were transferred to recovery solutions of various [K_o] at 38°C. The initial rate of K_i reaccumulation and steady state [K_i] were greater in solutions of higher [K_o]. Conversely for any time during recovery, the greater [K_o], the smaller the [K_i]:[K_o] ratio. When the strip was placed in the warm recovery solution it first contracted and then relaxed. The initial contraction was not relatable to [K_o] of the recovery solution but the subsequent relaxation was greater in rate and magnitude as [K_o] was greater. As the muscles recovered further they went into tonic contracture. As the [K_o] in the recovery solutions was greater these contractures occurred after shorter recovery times, and attained greater amplitude at a faster rate. Solution-switching experiments indicated a dependence of responses to electrical shocks on both the [K_i]:[K_o] ratio and [K_i]. Conclusions drawn were: (a) increased [K_i] increases contractility, (b) increased [K_i] increases the rate of relaxation, (c) excitability is decreased by too high or low a [K_i]: [K_o] ratio, and (d) the extent of tonic shortening depends on the [K_i]:[K_o] ratio.     

Calcium in the Control of Nitrogenase Activity in Vicia faba L. Root Nodules: Calcium Release from Symbiosomes and the Accompanying Decrease in Their Nitrogenase Activity Is Blocked by Verapamil

Treatment of root nodules or symbiosomes isolated from them with calcium chelator EGTA alone or together with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron-microscopic analysis revealed considerable calcium depletion of symbiosomes in the infected nodule cells treated with EGTA and A23187. Ca²⁺ channel blockers, verapamil and ruthenium red, inhibited EGTA-induced Ca²⁺ release from symbiosomes. In this case, NA insignificantly increased in the whole nodules and reached its initial level in symbiosomes. The experiments on isolated symbiosomes with arsenazo III, a Ca²⁺ indicator, demonstrated that verapamil inhibited Ca²⁺ release from them induced by valinomycin in the presence of K⁺ ions. These data suggest the presence on the peribacteroid membrane of a verapamil-sensitive transporter responsible for Ca²⁺ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of root nodules is discussed.     

Nitrogenase Activity in Relation to Intracellular Organisms in Sphagnum Mosses

Electron microscopic studies of Sphagnum lindbergii (Schimp.) and S. riparium (Ångstr.) have revealed the presence of intracellular organisms such as blue-green algae, green algae, bacteria and fungi. Nitrogenase activities of these Sphagnum mosses were found to be related mainly to the presence of intracellular Nostoc filaments. The appearance of nitrogen-fixing blue-green algae within bryophytes is thus not restricted to liverworts. The association is likely to be of ecological importance as it seems to occur in very acid habitats generally lacking blue-green algae. Possible interrelations between the moss, the blue-green algae and different types of bacteria are discussed.     

Effect of Temperature on Nitrogenase Activity in White Clover

Single, clonal plants of white clover were grown without inorganicnitrogen in four contrasting day/night temperature regimes,with a 12 h photoperiod, in controlled environments. Root andnodule respiration and acetylene reduction activity were measuredin a flow-through system during both day and night for plantsacclimated to day/night regimes of 23/18, 15/10 and 10/5 ?C.Similar measurements were made on plants acclimated to 20/15?C and stepwise at temperatures from 4 to 33 ?C. Peak rate of ethylene production, nitrogenase-linked respirationand basal root + nodule respiration increased approximatelylinearly from 5 to 23 ?C both in temperature-acclimated plantsand in plants exposed to varying measurement temperatures. Themeasured attributes did not vary significantly between day andnight. Temperatures above 23–25 ?C did not further enhancethe rate of ethylene production, which remained essentiallythe same up to the maximum measured temperature of 33 ?C. The measurements of nitrogenase-linked respiration between 5and 23 ?C, during both day and night, demonstrated a constant‘energetic cost’ of acetylene reduction of 2.9 µmolCO₂ µmol C₂H₄^–1,. Over the same temperature range,the approximate activation energy of acetylene reduction was60 kJ mol^–1. The integrated day plus night nitrogenase-linkedrespiration accounted for 13.4–16% of the plant‘snet shoot photosynthesis in a single diurnal period: there wasno significant effect of temperature between 5 and 23 ?C. Key words: Trifolium repens, white clover, temperature, N₂ fixation, respiration