Rates Of Nitrogenase Activity Research Articles

Consequences of Sporangial Development for Nodule Function in Root Nodules of Comptonia peregrina and Myrica gale

Frankia sp., the actinomycetous endophyte in nitrogen-fixing actinorhizal nodules, may differentiate two forms from its hyphae: vesicles and sporangia. In root nodules of Comptonia peregrina (L.) Coult. and Myrica gale L., sporangia may be either absent or present. Nitrogenase activity and symbiotic efficiency were contrasted in spore(+) and spore(-) nodules of these two host genera. Seedlings of C. peregrina nodulated with the spore(+) inoculum showed only 60% of the nitrogenase activity and 50% of the net size of their spore(-) counterparts after 12 weeks of culture. Measurements of acetylene reduction (i.e., nitrogenase activity) were coordinated with samplings of nodules for structural studies. Significant differences in acetylene reduction rates were discernible between spore(+) and spore(-) nodules commencing 4 weeks after nodulation, concomitant with the maturation of sporangia in the nodule. Spore(+) nodules ultimately reached less than half of the rate of nitrogenase activity of spore(-) nodules. Both types of nodules evolved only small amounts of molecular hydrogen, suggesting that both were equally efficient in recycling electrons lost to the reduction of hydrogen ions by nitrogenase. Respiratory cost of nitrogen fixation, expressed as the quotient of micromole CO(2) to micromole ethylene evolved by excised nodules, was significantly greater in spore(+) than in spore(-) nodules. M. gale spore(-) nodules showed variable effectivity, though all had low CO(2) to ethylene evolution ratios. M. gale spore(+) nodules resembled C. peregrina spore(+), with low effectivity and high respiratory cost for nitrogen fixation.

The pattern of in situ summer nitrogenase activity in terrestrial Nostoc commune from Stipa–Bouteloa grassland, southern Alberta

Field rates of nitrogenase activity by N. commune were followed over a 1-month summer period in Stipa–Bouteloa grassland of southern Alberta, using repeated short-term acetylene-reduction assays. Concurrent patterns of temperature, moisture, and light at the soil surface were continuously monitored. There is a rapid response of nitrogenase activity to both small precipitation events and dewfall hydration periods. Typically, thalli wet by thundershower activity in the previous evening showed a midday maximum of acetylene reduction of up to 5 nmol C2H4 mg−1 h−1, followed by a rapid cessation of activity as thalli desiccated below critical moisture levels. Over extended precipitation periods rates continue to climb throughout the day, gradually falling over the night period. Dry thalli frequently experience surface temperatures of up to 65 °C, without any discernable effects on rehydration recovery of nitrogenase activity. Equally, ground frosts early in the study period did not affect subsequent activity. Surface densities of up to 23.9 g m−2 were found for N. commune, with a mean cover of 4.3 g m−2. Mean cumulative nitrogen fixation over the 31-day field period was estimated to be 10.0 mg N m−2, although up to 77.3 mg N m−2 might be expected in localized microsites. The utilization of small rainfall events in large part accounts for the magnitude of observed nitrogenase activity. Although subsequent losses of fixed nitrogen through denitrification and volatilization are yet unknown, a significant proportion of nitrogen input to this grassland site must originate from asymbiotic biological fixation.

Enteromorpha and the cycling of nitrogen in a small estuary

The nitrogen relations of Enteromorpha spp. growing on intertidal mud flats have been examined over a twelve-month period. Nitrogen assimilation rates using 15N have been used to calculate the production of the alga and were between 0·046 and 0·217 mg NH 4 +N (g dry wt alga) −1 h −1 A considerable quantity of the alga was buried beneath the sediment over the growth season and was calculated to be equivalent to an input of up to 9·52 g N m −2 per month and 32 g N m −2 over one complete growth season. Based on carbon, this latter value represented an input of approximately 320 g C m −2 annually. Low rates of nitrogenase activity (acetylene reduction) were found to be associated with the Enteromorpha. The organisms responsible for the nitrogenase activity were probably heterotrophic bacteria but they did not contribute significant quantities of nitrogen to the alga.

Dinitrogen fixation by a mature Ceanothus velutinus (Dougl.) stand in the Western Oregon Cascades

Dinitrogen fixation was measured in a 17-year-old snowbrush, Ceanothus velutinus var. velutinus (Dougl.), stand in the Western Oregon Cascades. Diurnal and seasonal rates of nitrogenase activity were measured in the field using the C2H2 reduction technique. Snowbrush had a total biomass, estimated with equations developed, of 42 680 kg∙ha−1, including 750 kg∙ha−1 of nodule biomass. Snowbrush fixed N2 for approximately 240 days annually. Except during precipitation events or periods of low xylem pressure potentials, C2H2 reduction rates in the summer and fall were significantly correlated with soil temperature (R2 = 0.93**, n = 6). A diurnal variation in nitrogenase activity also was measured. The annual N2 fixation rate was estimated at approximately 101 kg N∙ha−1 (C2H2). Sustained periods of precipitation suppressed nitrogenase activity and reduced the estimate by about 19%. The annual N2 fixation rate is higher than previously reported for other mature stands and primarily is attributed to the maintenance of a large nodule biomass.

A Major Error in the Acetylene Reduction Assay: Decreases in Nodular Nitrogenase Activity Under Assay Conditions

Observations on both attached nodulated roots and detached nodules have revealed that nitrogenase activity in many legume species declined rapidly in the presence of acetylene, with a concurrent reduction in respiration. The reduction began within a few minutes of exposure to acetylene and continued for 30–60 min before a new steady-state was attained. A similar decline in H2 evolution and respiration was observed when N2 was replaced with argon or helium. This suggests that the decrease is linked to the cessation of ammonia production. Measurements of 15N2 uptake demonstrated that it is the pre-decline rather than final rate of ethylene production which represents the real rate of nitrogenase activity. The implications of these findings for the interpretation of acetylene reduction and hydrogen evolution data are considered.

Symbiosis, Nitrogen and Dry Matter Distribution in Chickpea (Cicer arietinum)

SUMMARYIn symbiotically-dependent chickpea, nodule mass and rates of nitrogenase activity increased when vegetative growth increased rapidly, but declined during fruiting. The specific activity of nodules declined as their mass rapidly increased. Nodule mass and rates of nitrogenase activity varied between genotypes, as did the point of maximum activity, which in some genotypes occurred before fruit growth dominated shoot growth. Current N2-fixation supplied most of the nitrogen for pod and seed growth, but in some genotypes mobilization of nitrogen from vegetative parts was evident. Seed attracted up to 50% of assimilated nitrogen; residual nitrogen was contained mainly in leaf and pod tissue, though in one genotype stem nitrogen was plentiful.

15N2 FIXATION AND MOLYBDENUM ENHANCEMENT OF ACETYLENE REDUCTION BY LOBARIA SPP.

SUMMARYThe lichens Lobaria pulmonaria and Lobaria oregana were assayed for 15N2 assimilation in order to confirm previous rates of nitrogenase activity measured by acetylene reduction. In addition, the effect of molybdenum on acetylene reduction activity was studied in both species. The molar conversion ratio (acetylene reduction rate divided by 15N2 fixation rate) was 4.27 for Lobaria pulmonaria and 4.78 for Lobaria oregana. The rate of 15N2 uptake decreased over time in both species. Addition of 1 p.p.m. molybdenum enhanced acetylene reduction by 180% in Lobaria pulmonaria and by 50% in Lobaria oregana. Optimum enhancement in Lobaria oregana was obtained with 1 p.p.m. molybdenum, while 10 p.p.m. may be inhibitory.

Ontogenetic Variation of Nitrogenase, Nitrate Reductase, and Glutamine Synthetase Activities in Oryza sativa

The relationship between the rates of nitrogenase, nitrate reductase, and glutamine synthetase activities, and plant ontogeny in rice (Oryza sativa L.), cultivar ;M9', grown in salt marsh sediment with and without nitrate treatment was studied. In both treatments, nitrogenase activity measured as the immediate linear rate of acetylene reduction by bacteria associated with the roots varied with plant age. In control plants, the nitrogenase activity developed during the vegetative stage, peaked during early reproductive growth and then declined. The application of 10 kilograms N per hectare as KNO(3) once every 2 weeks delayed the development of and decreased the nitrogenase activity. The nitrogenase activity in both treatments developed as leaf nitrate reductase activity declined. The per cent nitrogen of roots was negatively correlated with the rates of acetylene reduction during the life cycles of control and nitrate-treated plants. This suggests that the concentration of combined nitrogen in the plants controlled the development and rate of root-associated nitrogenase activity. During reproductive growth, no nitrate reductase activity was detected in the roots from either treatment. In control plants, the patterns of nitrogenase activity and glutamine synthetase activity in the roots were similar. Thus, rice roots have the potential to assimilate ammonia while fixing N(2). During the vegetative and early reproductive stages of growth, the development of maximal rates of nitrogenase activity coincided with an increase of total nitrogen of the plants in both treatments.

The association of N2-fixing bacteria with sea urchins

Dinitrogen fixation associated with bacteria in the gastrointestinal tract of sea urchins appears to be a widespread phenomenon: sea urchins from the tropics (Diadema antillarum, Echinometra lacunter, Tripneustes ventricosus), the temperature zone (Strongylocentrotus droebachiensis) and the arctic (S. droebachiensis) exhibited nitrogenase activity (C2H2 reduction). Pronounced seasonal variation was found in nitrogenase activity of temperate sea urchins feeding on kelp (Laminaria spp.) and eelgrass (Zostera marina). The mean monthly nitrogenase activity was inversely correlated with the nitrogen content of the sea urchin's food, which varied up to fivefold over the course of a year. The highest rate of nitrogenase activity recorded for a temperate sea urchin during the 14 month sampling period was 11.6μg N fixed g wet wt-1 d-1, with a yearly mean activity of 1.36 μg N fixed g wet wt-1 d-1. Studies with 15N confirmed the C2H2 reduction results and showed incorporation of microbially-fixed nitrogen into S. droebachiensis demonstrating that N2 fixation can be a source of N for the sea urchin. Laboratory experiments indicated that part of the sea urchin's (S. droebachiensis) normal gastrointestinal microflora is responsible for the observed nitrogenase activity.

PHYSIOLOGICAL‐ENVIRONMENTAL INTERACTIONS IN LICHENS XI. SNOWCOVER AND NITROGENASE ACTIVITY

SummaryThe influence of a long term snowcover on the rates of nitrogenase activity in the lichens Peltigera canina var. praetextata and P. canina var. rufescens has been investigated. In accordance with the existing literature, only very low rates of nitrogenase activity could be detected after several months of snowcover. These low rates of activity result from prolonged darkness rather than an inactivation of the nitrogenase enzyme by low temperature. The subsequent recovery of nitrogenase activity after snowmelt is very largely controlled by the presence of radiant energy with temperature playing a subordinate role.

Nitrogen Fixation by Photosynthetic Bacteria in Lowland Rice Culture

Propanil (3',4'-dichloropropionanilide) was a potent inhibitor of the nitrogenase activity of blue-green algae (cyanobacteria) in flooded soil, but the herbicide at comparable concentrations was not toxic to rice, protozoa, and nitrogen-fixing bacteria. Ethanol-amended flooded soils treated with propanil exhibited higher rates of nitrogenase activity than those not treated with the herbicide. The enhanced nitrogenase activity in propanil-treated soils was associated with a rise in the population of purple sulfur bacteria, especially of cells resembling Chromatium and Thiospirillum. By employing propanil and a means of excluding light from the floodwater to prevent the development of phototrophs during rice growth under lowland conditions, the relative activities of blue-green algae, photosynthetic bacteria, and the rhizosphere microflora were determined. The results suggest that the potential contribution of photosynthetic bacteria may be quite high.

Some physiological‐biochemical characteristics of planktonic blue‐green algae during bloom formation in three Salopian meres

SUMMARY. Levels of cellular orthophosphate concentration, alkaline phosphatase activity, photosynthesis and nitrogenase activity of natural populations of blue‐green algae collected during the summer of 1976 from the surface waters of three eutrophic lakes were determined under controlled conditions. Intracellular concentrations of surplus stored phosphorus remained high and alkaline phosphatase activity remained low during the formation and decline of the surface bloom, which indicated that available phosphorus does not become limiting to growth. In contrast, declining rates of nitrogenase activity and net photosynthesis were recorded, particularly in those blooms of shorter duration. An increase in numbers of bacteria during the summer may account for the observed fall of net photosynthetic rates, since gross rates did not change as markedly. There was appreciable nitrogenase activity proceeding and during the differentiation of akinetes, and the cellular C/N atomic ratio of sporulating populations was lower than in non‐sporulating algae. It is suggested that the onset of sporulation in surface blooms is initiated by an imbalance of the normal vegetative growth of the algal cells and of their carbon and nitrogen metabolism.

Nitrogen Fixation in Relation to Photosynthesis in Alnus glutinosa

Clonal lines of Alnus glutinosa (L.) Gaertn., grown in combination with a spore-negative North American endophyte, were studied to determine relationships between nitrogen fixation and photosynthesis in nitrogen-free culture and to determine the effect of host-plant genotype on symbiotic growth. The correlations between leaf area and acetylene reduction and between photosynthetic rate and nitrogen content in a nodulated plant were significant. Growth among clonal alder lines did not differ 8 wk after inoculation with a North American crushed-nodule preparation. Significant differences did occur after 8 wk, however, among alder clones grown on combined nitrogen. Genetic variation in the host as well as the source of the endophyte seems to condition the related rates of nitrogenase activity and photosynthesis in black alder.

Biological dinitrogen fixation (acetylene reduction) associated with Florida mangroves.

Biological dinitrogen fixation in mangrove communities of the Tampa Bay region of South Florida was investigated using the acetylene reduction technique. Low rates of acetylene reduction (0.01 to 1.84 nmol of C(2)H(4)/g [wet weight] per h) were associated with plant-free sediments, while plant-associated sediments gave rise to slightly higher rates. Activity in sediments increased greatly upon the addition of various carbon sources, indicating an energy limitation for nitrogenase (C(2)H(2)) activity. In situ determinations of dinitrogen fixation in sediments also indicated low rates and exhibited a similar response to glucose amendment. Litter from the green macroalga, Ulva spp., mangrove leaves, and sea grass also gave rise to significant rates of acetylene reduction. Higher rates of nitrogenase activity (15 to 53 nmol of C(2)H(4)/g [wet weight] per h were associated with washed excised roots of three Florida mangrove species [Rhizophora mangle L., Avicennia germinans (L) Stern, and Laguncularia racemosa Gaertn.] as well as with isolated root systems of intact plants (11 to 58 mug of N/g [dry weight] per h). Following a short lag period, root-associated activity was linear and did not exhibit a marked response to glucose amendment. It appears that dinitrogen-fixing bacteria in the mangrove rhizoplane are able to use root exudates and/or sloughed cell debris as energy sources for dinitrogen fixation.

Discovering the Role of Lichens in the Nitrogen Cycle in Boreal-Arctic Ecosystems

Discovering the Role of Lichens in the Nitrogen Cycle in Boreal-Arctic Ecosystems

ATP AND NITROGENASE ACTIVITY IN NITROGEN‐FIXING HETEROCYSTOUS BLUE‐GREEN ALGAE

SUMMARYThe relationship between ATP and nitrogenase activity has been investigated using the obligate photoautotroph Anabaena cylindrica and Anabaenopsis circularis strain 6720, a photoautotroph which can also use exogenously supplied carbon. In the light photophosphorylation supports maximum observed rates of nitrogenase activity (0.60 and 1.2 μmoles C2H4 mg chl a−1 min−1 under aerobic and microaerobic conditions respectively). The ATP pools sustained under both conditions are similar at 160–200 μmoles mg chl a−1. Oxidative phosphorylation and substrate level phosphorylation are unimportant in supporting nitrogenase activity in the light. Cyclic photophosphorylation can supply all the necessary ATP provided fixed carbon compounds from photosynthetic CO2 fixation (Anabaena) or supplied exogenously (0.2%, w/v, fructose) (Anabaenopsis) are available. When the algae are placed in the dark nitrogenase activity declines. In Anabaenopsis, after an initial rapid drop, nitrogenase activity can be sustained in the presence of exogenous carbohydrate in the dark at up to 70% of the light rate under air, but not under anaerobic conditions. This decreased nitrogenase activity in the dark is not due to a lack of nitrogenase synthesis or of penetration of fixed carbon into the cells, but appears to be due to an inherent inability of dark metabolism to generate sufficient ATP and/or reductant for maximum nitrogenase activity. The ATP pool can be sustained at maximum levels when photophosphorylation, cyclic photophosphorylation, and oxidative phosphorylation operate individually, and to a lesser extent when substrate‐level phosphorylation operates. No consistent correlations were found between the ATP pool and nitrogenase activity, except under dark anaerobic conditions where there was a positive correlation. The data suggest either that the ATP pool which supports nitrogenase activity is distinct from that which supplies ATP for general filament metabolism, and/or that the supply of ATP is not the most critical short‐term factor in controlling nitrogenase activity in Anabaena and Anabaenopsis.

PHYSIOLOGICAL‐ENVIRONMENTAL INTERACTIONS IN LICHENS

SUMMARYThe recovery rate of nitrogenase activity in Peltigera polydactyla, is shown to be rapid after short drought periods but takes progressively longer as the drought period is extended. After more extreme levels of dehydration achieved using calcium chloride, recovery of nitrogenase activity is considerably slower. The importance of adequate pretreatment of material collected from the field after exposure to unknown periods and levels of drought stress is emphasized.

THE CYANOPHYTE‐HEPATIC SYMBIOSIS I. MORPHOLOGY AND PHYSIOLOGY

SUMMARYThe Anthoceros punctatus‐Nostoc and Blasia pusilla‐Nostoc symbioses were investigated. In both associations the Nostoc colonies develop in mucilaginous cavities on the undersurface of the gametophyte. The sporophytes of Anthoceros have no Nostoc colonies; no sporophytes of Blasia were found. The symbiotic algae have been identified as Nostoc sphaericum ex. Born et Flah. The heterocyst frequency of the free‐living isolates is 3–6% but this increases to 30% (Blasia) and 43% (Anthoceros) when the algae are growing symbiotically. On infecting alga‐free gametophytes of Blasia pusilla with Nostoc, algal colonies develop in the cavities within 72 h. The developing Nostoc colonies stretch the cells of the cavity wall and there also arise from a point on the host cavity wall, usually opposite the cavity pore, septate, branched, filamentous protrusions, which increase the surface area of contact between phycobiont and host by about 30% within 4 weeks of colony formation. Such outgrowths may facilitate interchange of metabolites between the alga and liverwort. The Nostoc phycobiont of Anthoceros may infect Blasia and vice‐versa. A strain of Nostoc punctiforme isolated from Gunnera also infects Blasia, but four other Nostoc isolates, including one from cycad root nodules, do not. Five other heterocystous algae, five non‐heterocystous filamentous algae and one unicellular alga tested do not infect Blasia. The nitrogen contents of thalli with Nostoc colonies are significantly higher than those treated with algae which do not develop colonies. The symbiotic algae differ physiologically and biochemically from free‐living algae. They show higher rates of nitrogenase activity (acetylene reduction assay), are depleted of nitrogen‐storing phycobilin pigments and structured granules and although metabolically active do not evolve O2 or fix CO2 photosynthetically, but do have polyhedral bodies which contain the key CO2‐fixing enzyme ribulose‐l,5‐diphosphate carboxylase. The Blasia symbiosis grows well and remains established in the pH range 4–8, at low levels of combined nitrogen (<100 mg I−1 of nitrate‐nitrogen) but not at high levels (100–1000 mg I−1), in the temperature range 10–25°C, and under moist but not waterlogged or desiccated conditions.

Interactions Between Growth, Phosphate Content and Nitrogen Fixation in Mycorrhizal and Non-Mycorrhizal Medicago sativa

Nodulated Medicago sativa cv. Europe plants, both non-mycorrhizal and mycorrhizal (inoculated with Glomus mosseae), were grown in sand or soil with a range of phosphate levels. The following parameters were measured: intensity of mycorrhizal infection, intensity of nodulation, growth, phosphate content, nitrogenase activity (acetylene reduction) and nitrogen content. Both nodulation and mycorrhizal infection had occurred within 2 weeks of inoculation with the appropriate endophytes. Major increases in dry weight of mycorrhizal plants were not apparent until approximately 10 weeks from inoculation. However, mycorrhizal plants showed more extensive nodulation, coupled with higher rates of nitrogenase activity from 2 weeks onwards, and at the final (12-week) harvest had higher values of % N. Phosphate content of mycorrhizal plants (�g P/g dry wt) was also greater than non-mycorrhizal plants at 7 weeks. By 10 or 12 weeks, when significant mycorrhizal enhancement of growth was apparent, the total nitrogen and total phosphorus per mycorrhizal plant were also higher, but nitrogenase activity and phosphate content measured on a dry weight basis showed no significant differences between mycorrhizal and non-mycorrhizal plants. It is clear that mycorrhizal enhancement of phosphate uptake and nitrogen fixation precedes any effect on growth and the results indicate the importance of the time factor in the development of this tripartite symbiosis between legume, Rhizobium and mycorrhizal fungus.

Leghaemoglobin and the Supply of O2 to Nitrogen-fixing Root Nodule Bacteroids: Studies of an Experimental System with No Gas Phase

SUMMARY: The effects of purified oxyleghaemoglobin added to suspensions of bacteroids prepared anaerobically from soybean root nodules, were studied in terms of uptake of dissolved O2, nitrogenase activity and dissolved O2 concentration, in the absence of a gas phase. Leghaemoglobin allowed maximum rates of O2 uptake to continue to a much lower range of concentrations of free dissolved O2 than in the absence of the protein. This effect was diminished when the leghaemoglobin concentration was less than about 50 μ m. Nitrogenase activity at a given O2 concentration was not increased by raising the leghaemoglobin concentration above about 50 μ m. The fractional oxygenation of leghaemoglobin giving half the maximal O2 consumption rate by bacteroids was usually about 0·2; with limiting leghaemoglobin concentrations it was higher. Rates of nitrogenase activity were invariably greater during periods when the discharge of O2 from oxyleghaemoglobin was occurring at the maximum rate, than when similar maximum O2 uptake rates were being supported by higher concentrations of free dissolved O2. When myoglobin was used as the O2 carrier protein in similar experiments, enhanced nitrogenase activity also accompanied the maximum rate of discharge from the carrier. This occurred at about four times the concentration of free dissolved O2 at which it occurred when leghaemoglobin was the carrier. These results are discussed in relation to current theories about the mechanism of leghaemoglobin action.