A non-destructive field assay for soybean nitrogen fixation by acetylene reduction

Summary A system for employing open-ended root chambers to measurein situ acetylene reduction rates under field conditions is described. Gas mixtures containing about 2 mbar acetylene were continuously flowed through the chambers providing a continuous record of acetylene reduction. These chambers have been used to measure acetylene reduction rates of soybeans during three growing seasons. The system has proved to be reliable with a high degree of precision. The large amount of plant-to-plant variability observed in N₂ fixation research has been confirmed by the data collected with this system. However, such variability in physiological studies can be reduced by using a non-destructive system to compare the response of an individual plant with its rates before treatment.     

Non-destructive semi-quantitative determination of acetylene reduction in the field

Summary Direct injection of acetylene into soil around plant roots, followed by determination of ethylene/acetylene ratios in the soil atmosphere has been tested as a rapid, non-destructive method of estimating acetylene reducing activity. In pots of artificial media as well as in field soil, the ratios determined within 10 min. after injection were significantly correlated with the rates of acetylenedependent ethylene production in detached roots. The method may be useful in preliminary screening of large numbers of plant-bacteria combinations.     

Dark anaerobic nitrogen fixation (acetylene reduction) in the cyanobacterium Oscillatoria sp.

The filamentous, non-heterocytous, nitrogen-fixing cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) showed cycling of acetylene reduction in light-dark cycles. Under aerobic conditions nitrogenase activity is exclusively present during the dark period. However, if anaerobic conditions were applied during the dark period, two activity maxima were observed. A relatively small activity peak occurred during the first few hours of the dark period and a high peak as soon as the light was switched on. A low activity remained during the second half of the dark period. This pattern of acetylene reduction in Oscillatoria agrees well with the field data on nitrogen fixation [Stal, L.J. and Krumbein, W.E. (1984), Mar. Biol. 82, 217–224].     

Flavin-mediated reduction of ferric leghemoglobin from soybean nodules

The reduction of ferric leghemoglobin (Lb³⁺) from soybean (Glycine max (L.) Merr.) nodules by riboflavin, FMN and FAD in the presence of NAD(P)H was studied in vitro. The system NAD(P)H + flavin reduced Lb³⁺ to oxyferrous (Lb²⁺ · O₂) or deoxyferrous (Lb²⁺) leghemoglobin in aerobic or anaerobic conditions, respectively. In the absence of O₂ the reaction was faster and more effective (i.e. less NAD(P)H oxidized per mole Lb³⁺ reduced) than in the presence of O₂; this phenomenon was probably because O₂ competes with Lb³⁺ for reductant, thus generating activated O₂ species. The flavin-mediated reduction of Lb³⁺ did not entail production of superoxide or peroxide, indicating that NAD(P)H-reduced flavins were able to reduce Lb³⁺ directly. The NAD(P)H + flavin system also reduced the complexes Lb³⁺ · nicotinate and Lb³⁺ · acetate to Lb²⁺ · O₂, Lb²⁺ or Lb²⁺ · nicotinate, depending on the concentrations of ligands and of O₂. In the presence of 200 M nitrite most Lb remained as Lb³⁺ in aerobic conditions but the nitrosyl complex (Lb²⁺ · NO) was generated in anaerobic conditions. The above-mentioned characteristics of the NAD(P)H + flavin system, coupled with its effectiveness in reducing Lb³⁺ at physiological levels of NAD(P)H and flavins in soybean nodules, indicate that this mechanism may be especially important for reducing Lb³⁺ in vivo.Abbreviations and Terminology FLbR ferric leghemoglobin reductase - Hb²⁺ /Hb³⁺ hemoglobin containing Fe²⁺ /Fe²⁺ - Lb²⁺ /Lb³⁺ leghemoglobin containing Fe²⁺ /Fe³⁺ - Lb³⁺ · nicotinate/acetate Lb in which nicotinate or acetate are complexed to Lb³⁺ - Lb²⁺ · O₂/CO/NO/nicotinate Lb in which O₂, CO, NO or nicotinate are complexed to Lb²⁺ - Rfl riboflavin - SOD superoxide dismutase (EC 1.15.1.1) Published as Paper No. 9237, Journal Series, Nebraska Agricultural Research DivisionWe thank M.B. Crusellas for his skillful drawings. M. Becana thanks the Spanish Ministry of Education and Science/Fulbright Commission for financial support.     

Heterotrophic nitrogen fixation (acetylene reduction) associated to flooded rice: A modified measurement technique in the field

A modifiedin situ technique for measuring heterotrophic nitrogen fixing (acetylene reducing) activity associated to rice is proposed. Ethylene evolution rates measured in opaque cylinders covering the stems of rice plants which have been cut 10 cm over the water level were found independent of the diurnal cycle. Cutting of the leaves resulted in decreased variation between plants and suppression of the acceleration of ethylene evolution rate after 12 h incubation as compared to intact plants. In both systems ethylene evolved was swept by a current of methane and the molar ratio between methane and ethylene was stabilized after 12 h. Methane evolution rates remained stable during 12 h and more than 24 h in whole plants and cut plants respectively. It is suggested that alteration in the active gas transport system after 12 h incubation under 10% acetylene may lead to erroneous evaluation of the actual ethylene production in the root's environment. The average values of ethylene evolution rates by cut plants between 12 and 24 h of incubation may be used for comparative studies of nitrogen fixing activity associated to flooded rice.     

Microcosm investigation of growth and phytoremediation potential of Azolla japonica along nitrogen gradients

Although Azolla species are among the most promising plants for use in phytoremediation, more studies on their growth and nitrogen (N) uptake along the N gradients of growing media are required. In this study, N concentration-dependent growth in growing media and phosphorus (P) and N accumulation by Azolla japonica were studied by estimating direct N uptake from media by molybdenum-iron proteins. The doubling time of A. japonica was less than a week, regardless of the N concentration (0, 5, and 25 mg N/L) present in the growth media, indicating that this plant is suitable for remediation. Plants showed a high uptake of P, probably via plant-bacteria symbiosis, indicating their potential for effective P remediation. A. japonica also showed more than 4% N content regardless of the treatment and accumulated more than 40 mg of N per microcosm in 3 weeks. iron and molybdenum levels in plants were strongly associated with N fixation, and N uptake from media was estimated to be more than 25 mg per microcosm in 3 weeks, indicating that A. japonica has N remediation potential. As A. japonica is a rapidly growing plant, capable of efficient P and N remediation, it has great potential for use in phytoremediation of nutrient-enriched waters such as agricultural or urban wastewater and eutrophicated aquatic ecosystems.     

Ferrous iron is transported across the peribacteroid membrane of soybean nodules

Symbiosomes and bacteroids isolated from soybean (Glycine max Merr.) nodules are able to take up ferrous iron. This uptake activity was completely abolished in the presence of ferrous-iron chelators. The kinetics of uptake were characterized by initially high rates of iron internalization, but no saturation was observed with increasing iron concentration. This process does not appear to involve the ferric reductase of the peribacteroid membrane. The transport of ferrous iron was inhibited by other transition metals, particularly copper. Ferrous iron was taken up by symbiosomes more efficiently than the ferric form. This indicates that the iron transport from the plant host cell to the microsymbiont in vivo may occur mainly as the ferrous form. Received: 11 February 1998 / Accepted: 29 May 1998     

Discrimination of nitrogen isotopes during absorption of ammonium and nitrate at different nitrogen concentrations by rice (Oryza sativa L.) plants

Studies of uptake of ionic sources of N by two hydroponically grown rice (Oryza sativa L.) cultivars (paddy‐field‐adapted Koshihikari and dryland‐adapted Kanto 168) showed that the magnitude of the nitrogen isotope fractionation (?) for uptake of NH₄⁺ depended on the concentrations of NH₄⁺ and cultivar (averaging –6·1‰ for Koshihikari and –12·0‰ for Kanto 168 at concentrations from 40 to 200 mmol m^?3 and, respectively, –13·4 and –28·9‰ for the two cultivars at concentrations from 0·5 to 4 mol m^?3). In contrast, the ? for uptake of NO₃^? in similar experiments was almost insensitive to the N concentration, falling within a much narrower range (+3·2‰ to –0·9‰ for Koshihikari and –0·9‰ to –5·1‰ for Kanto 168 over NO₃^? concentrations from 0·04 to 2 mol m^?3). From longer term experiments in which Norin 8 and its nitrate‐reductase deficient mutant M819 were grown with 2 or 8 mol m^?3 NO₃^? for 30 d, it was concluded that the small concentration‐independent isotopic fractionation during absorption of this ion was not related to nitrate reductase activity.     

外源NH_4~+对穗花狐尾藻根系形态和养分吸收的影响

在温室内进行静态实验,以原沉积物(CK)和分别添加0.24%和0.48%氯化铵(SN1和SN2)的沉积物作为底质培养沉水植物,探讨了高NH4+环境中穗花狐尾藻的根系形态特征及营养物质积累与干物质分配策略。结果表明,穗花狐尾藻主根直径(0.432-0.518mm)与主根/侧根比率(1.50-4.39)均随着沉积物NH4+含量升高呈现增大趋势,但单株主根总长(31.64-171.67cm)则在高NH4+环境中显著变短;其中,SN2处理中穗花狐尾藻主根数量(8.17条/株)显著低于SN1(14.67条/株)和CK(14.33条/株)处理,而SN1与CK处理之间差异不显著。SN1和SN2处理中穗花狐尾藻植株全氮含量(55.98和55.19mg/g)均显著高于CK(42.89mg/g)处理,而SN1和SN2处理中穗花狐尾藻植株全磷含量(1.63和1.53mg/g)则比CK处理中(3.71mg/g)显著降低。穗花狐尾藻植株干物质积累量(168.17-405.81mg/株)和全磷积累量(0.25-1.51mg/株)均随沉积物NH4+含量升高而显著下降,但植株全氮积累量(9.12-21.08mg/株)则表现为:SN1CKSN2。全氮和全磷在叶片中的分配率都随着沉积物NH+含量升高而显著降低,而在根系和茎秆中分配率则显著增加。     

Interspecies differences in the preference of ammonium and nitrate in vascular plants

Three solution experiments were performed to test the importance of NH ₄ ⁺ versus NO ₃ ^- +NH ₄ ⁺ to growth of 23 wild-forest and open-land species, using field-relevant soil solution concentrations at pH 4.5. At N concentrations of 1–200 M growth increased with increasing N supply in Carex pilulifera, Deschampsia flexuosa, Elymus caninus and Bromus benekenii. Geum urbanum was the most N demanding species and had little growth below 200 M. The preference for NH ₄ ⁺ or NO ₃ ^- +NH ₄ ⁺ was tested also at pH 4.0; no antagonism was found between NH ₄ ⁺ and H⁺, as indicated by similar relative growth in both of the N treatments at both pH levels. Growth in solution with NH ₄ ⁺ relative to NO ₃ ^- +NH ₄ ⁺ , 200 M, was negatively related to the mean pH of the field occurrence of the species tested; acid-tolerant species grew equally well with only NH ₄ ⁺ as with NO ₃ ^- +NH ₄ ⁺ (Oxalis acetosella, Carex pilulifera, Festuca gigantea, Poa nemoralis, Deschampsia flexuosa, Stellaria holostea, Rumex acetosella), while species of less acid soils were favoured by NO ₃ ^- +NH ₄ ⁺ (Urtica dioica, Ficaria verna, Melandrium rubrum, Aegopodium podagraria, Geum urbanum, Bromus benekenii, Sanguisorba minor, Melica ciliata, Silene rupestris, Viscaria vulgaris, Plantago lanceolata). Intermediate species were Convallaria majalis, Elymus caninus, Hordelymus europaeus and Milium effusum. No antagonism between NH ₄ ⁺ and Ca²⁺, Mg²⁺ and K⁺ was indicated by the total uptake of the elements during the experiment.     

Effect of lime additions to acid strip-mine spoil on survival,growth and nitrogen fixation (acetylene reduction) of several woody legume and actinomycete-nodulated species

Summary An acid mine spoil in Southern Indiana was amended with lime (CaCO₃) (0.0, 12.5, 25 and 39t/ha) and planted withElaegnus umbellata Thunb.,Alnus glutinosa Gaertn.,Robinia pseudoacacia L.,Robinia fertilis Ashe, Arnot,Myrica pensylvania Lois,Caragana arborescens L. andShepherdia argentea Nutt. Survival and soil data were collected periodically and plants were harvested 15 months after planting. Nodule and top dry weights were determined and acetylene reduction assays performed on the nodules.Addition of lime caused significant increases in pH, and 39 t/ha of lime were required to maintain a pH above 5.5. Survival of plant material was greatest at the highest lime addition, although response of individual species varied.Elaeagnus umbellata, R. pseudoacacia, R. fertilis Arnot, andA. glutinosa appeared more tolerant of the harsh conditions. OnlyC. arborescens showed a linear increase in top dry weight due to lime addition.Alnus glutinosa andS. argentea achieved statistically the same growth regardless of pH, andR. fertilis Arnot andE. umbellata did not increase in top dry weight above an addition of 25 t/ha.Robinia pseudoacacia achieved maximum top dry weight at 25 t/ha, whereasM. pensylvanica growth declined with increasing pH. Nodule dry weights increased with increasing pH; however,S. argentea showed greater nodule dry weights at lower lime levels. Acetylene reduction rates increased with lime addition.Elaegnus umbellata did not respond above 25 t/ha lime, whereasA. glutinosa did not show an increase until this point.     

Relationship between porewater organic carbon content, sulphate reduction and nitrogen fixation (acetylene reduction) in the rhizosphere of Zostera noltii

Depth profiles of nitrogen fixation (acetylene reduction), sulphate reduction, NH ₄ ⁺ concentration and porewater volatile fatty acids concentrations were measured in Zostera noltii colonised sediments in the Bassin d'Arcachon, France in March 1994. Acetylene reduction activity (ARA) was detectable throughout sediment profiles. Addition of sodium molybdate (20 mmol l^–1) a specific inhibitor of sulphate reduction to slurries inhibited ARA by >75% inferring that sulphate-reducing bacteria (SRB) were the dominant component of the nitrogen fixing microflora. The peak of ARA was coincident with that of sulphate reduction and a relatively constant relationship of 40 mole sulphate reduced per mole acetylene reduced was recorded throughout the profiles. From this ratio it was calculated that at least 17% of the ATP yield from sulphate reduction would be required to support the measured rates of nitrogen fixation (acetylene reduction).Acetate was the dominant constituent of the porewater volatile fatty acids pool, accounting for >90% of the total pool as measured by HPLC. Concentrations of porewater acetate recorded by HPLC were compared with those measured using an enzymatic technique and these data indicate that approximately 10% of the total porewater acetate pool was not available to microbial metabolism. Profiles of porewater acetate concentrations measured by both techniques were similar to those recorded for both ARA and sulphate reduction and thus acetate oxidation may fuel these activities.     

Effects of delayed soil and foliar N fertilization on yield and N2 fixation of soybean

Summary Field experiments were conducted to determine the effects of the amount, time and method of fertilizer N application on the efficiency of N uptake, N₂ fixatio and yield of soybean. Soil and foliar fertilizer N, applied during the pod-filling stage were absorbed by plants with equal and high efficiency, compared to an appreciably lower utilization efficiency for N applied before seedling emergence. These results reveal that the soybean roots were active in N uptake during these late stages of growth. Nitrogen fertilization during pod-filling resulted in significant yield increases over the control treatment which received an early application of 20 Kg N/ha. Seed yield increases were, however, more pronounced than total dry matter yield, and virtually all of the late-applied N was translocated into the pods. Nitrogen fixation in soybean was not influenced by the application of 40 kg N/ha to plants as soil or foliar N during the pod-filling stage. However, 80 kg N/ha supplied during pod-filling as 40 kg soil plus 40 kg foliar N/ha significantly reduced the amount of N₂ fixed. The results obtained in these studies suggest that inadequate N supply during pod-filling limited soybean yields, and that by the judicious application of fertilizer N during the late stages of growth, it was possible to enhance soybean yields without necessarily inhibiting N₂ fixation.     

Ineffective utilization of nitrate by soybean during pod fill

The relative effectiveness of nitrate, allantoin, or nitrate plus allantoin as sources of nitrogen for the indeterminate soybean plant [ Glycine max (L.) Merr cv. Harper] was studied throughout vegetative and reproductive growth. All plants were provided with 3.0 m M nitrogen and were grown hydroponically in growth chambers. During vegetative and early reproductive growth, plants given nitrate or nitrate plus allantoin grew faster than plants provided allantoin only. However, during pod fill, plants provided with allantoin or allantoin plus nitrate gained weight more rapidly than plants receiving just nitrate. More importantly, at maturity plants that had been provided with allantoin or allantoin plus nitrate during pod fill were 30% heavier in total dry weight, 50% higher in nitrogen content, and 50% higher in seed yield than plants that had received just nitrate. At full bloom, all plants were inoculated with the same culture of Bradyrhizobium japonicum , and twice each week throughout pod fill each plant was assayed for nitrogen fixation (acetylene reduction). Correlation coefficients obtained by linear regression analysis show a strong positive correlation between the measured rate of nitrogen fixation and maximum plant fresh weight (r = 0.83), total plant nitrogen (r = 0.81), or seed yield (r = 0.76). The fact that nitrogen fixation during pod fill stimulates plant growth and seed yield, coupled with the facts that nitrate blocks nodulation and is not used efficiently during pod fill by the soybean plant, may explain why seed yield of field-grown soybeans usually does not respond to added fertilizer nitrogen. Thus, it is suggested that enhanced nitrogen fixation may be the key factor in improving soybean seed yield.     

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy (Oryza sativa L.)

Summary An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.     

Absence of nitrogen fixation (acetylene reduction) by procaryotes in nectar of Banksias

Summary An earlier hypothesis that blue-green algae in the nectar ofBanksia telmatiaea contribute to the nitrogen economy of the host by fixing N₂ was tested. Field and laboratory experiments failed to demonstrate C₂H₄ production in C₂H₂-treated containers over extended periods. Soil N was not higher at the end of the flowering season and plants in which flower heads were removed prior to nectar production did not contain less N than the controls.     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

Free amino acid, ammonium and nitrate concentrations in soil solutions of a grazed coastal marsh in relation to plant growth

Soluble free amino acids, ammonium and nitrate ions as sources of nitrogen for plant growth were measured in soils of a coastal marsh grazed by snow geese in Manitoba, Canada. Amounts of nitrogen, primarily ammonium ions, increased in the latter half of the growing season and over winter, but fell to low values early in the growing season. Free amino acid concentrations relative to ammonium concentrations were highest during the period of rapid plant growth in early summer, especially in soils in the intertidal zone, where the median ratio of amino acid nitrogen to ammonium nitrogen was 0·36 and amino acid concentrations exceeded those of ammonium ions in 24% of samples. Amino acid profiles, which were dominated by alanine, proline and glutamic acid, were similar to goose faecal profiles. In a continuous flow hydroponic experiment conducted in the field, growth of the salt‐marsh grass, Puccinellia phryganodes, on glycine was similar to growth on ammonium ions at an equivalent concentration of nitrogen. When supplies of soil inorganic nitrogen are low, amino acids represent a potentially important source of nitrogen for the re‐growth of plants grazed by geese and amino acid uptake may be as high as 57% that of ammonium ions.